Method for inactivating pathogens in blood using photoactivation of 4&#39;-primary amino-substituted psoralens

ABSTRACT

Psoralen compounds are synthesized which have substitutions on the 4, 4&#39;, 5&#39;, and 8 positions of the psoralen, which permit enhanced binding to nucleic acid of pathogens. Higher psoralen binding levels and lower mutagenicity are described, resulting in safer, more efficient, and reliable inactivation of pathogens in blood products. The invention contemplates inactivation methods using the new psoralens which do not compromise the function of blood products for transfusion.

The present application is a continuation in part of U.S. applicationSer. No. 08/212,113, filed Mar. 11, 1994 now abandoned, which is acontinuation in part of U.S. application Ser. No. 08/083,459, filed Jun.28, 1993 now U.S. Pat. No. 5,399,719.

FIELD OF THE INVENTION

The present invention provides methods of using new and known compoundsto inactivate bacteria in health related products to be used in vivo andin vitro, and particularly, in blood products and blood products insynthetic media.

BACKGROUND

Pathogen contamination within the blood supply remains an importantmedical problem throughout the world. Although improved testing methodsfor hepatitis B (HBV), hepatitis C (HCV), and Human ImmunodeficiencyVirus (HIV) have markedly reduced the incidence of transfusionassociated diseases, the public is losing trust in the safety of theblood supply due to publicity of cases of transfusion relatedtransmission of these viruses.

For example, the recent introduction of a blood test for HCV will reducetransmission of this virus; however, it has a sensitivity of only 67%for detection of probable infectious blood units. HCV is responsible for90% of transfusion associated hepatitis. Melnick, J. L., abstracts ofVirological Safety Aspects of Plasma, Cannes, Nov. 3-6 (1992) (page 9).It is estimated that, with the test in place, the risk of infection is 1out of 3300 units transfused.

Similarly, while more sensitive seriological assays are in place forHIV-1 and HBV, these agents can nonetheless be transmitted byseronegative blood donors. International Forum: Vox Sang 32:346 (1977).Ward, J. W., et al., N. Engl. J. Med., 318:473 (1988). Up to 10% oftotal transfusion-related hepatitis and 25% of severe icteric cases aredue to the HBV transmitted by hepatitis B surface antigen (HBasAg)negative donors. To date, fifteen cases of transfusion-associated HIVinfections have been reported by the Center for Disease Control (CDC)among recipients of blood pre tested negative for antibody to HIV-1.

Furthermore, other viral, bacterial, and agents are not routinely testedfor, and remain a potential threat to transfusion safety. Schmunis, G.A., Transfusion 31:547-557 (1992). In addition, testing will not insurethe safety of the blood supply against future unknown pathogens that mayenter the donor population resulting in transfusion associatedtransmission before sensitive tests can be implemented.

Even if seroconversion tests were a sufficient screen, they may not bepractical in application. For example, Cytomegalovirus (CMV--a herpesvirus) and parvo B19 virus in humans are common. When they occur inhealthy, immunocompetent adults, they nearly always result inasymptomatic seroconversion. Because such a large part of the populationis seropositive, exclusion of positive units would result in substantiallimitation of the blood supply.

An alternative approach to eliminate transmission of viral diseasesthrough blood products is to develop a means to inactivate pathogens intransfusion products. Development of an effective technology toinactivate infectious pathogens in blood products offers the potentialto improve the safety of the blood supply, and perhaps to slow theintroduction of new tests, such as the recently introduced HIV-2 test,for low frequency pathogens. Ultimately, decontamination technologycould significantly reduce the cost of blood products and increase theavailability of scarce blood products.

To be useful, such an inactivation method i) must not adversely effectthe function for which the blood product is transfused, ii) mustthoroughly inactivate existing pathogens in the blood product, and iii)must not adversely effect the recipients of the blood product. Severalmethods have been reported for the inactivation or elimination of viralagents in erythrocyte-free blood products. However, most of thesetechniques are completely incompatible with maintenance of the functionof platelets, an important blood product. Examples of these techniquesare: heat (Hilfenhous, J., et al., J. Biol. Std. 70:589 (1987)),solvent/detergent treatment (Horowitz, B., et al., Transfusion 25:516(1985)), gamma-irradiation (Moroff, G., et al., Transfusion 26:453(1986)), ultraviolet (UV) radiation combined with beta propriolactone,(Prince A. M., et al., Reviews of Infect. Diseases 5: 92-107 (1983)),visible laser light in combination with hematoporphyrins (Matthews J.L., et al., Transfusion 28: 81-83 (1988); North J., et al., Transfusion32: 121-128 (1992)), use of the photoactive dyes aluminumphthalocyananine and merocyanine 540 (Sieber F., et al., Blood 73:345-350 (1989); Rywkin S., et al., Blood 78(Suppl 1): 352a (Abstract)(1991)) or UV alone (Proudouz, K. N., et al., Blood 70:589 (1987)).

Other methods inactivate viral agents by treatment with furocoumarins,such as psoralens, in the presence of ultra-violet light. Psoralens aretricyclic compounds formed by the linear fusion of a furan ring with acoumarin. Psoralens can intercalate between the base pairs ofdouble-stranded nucleic acids, forming covalent adducts to pyrimidinebases upon absorption of long wave ultraviolet light (UVA). G. D. Ciminoet al., Ann. Rev. Biochem. 54:1151 (1985); Hearst et al., Quart. Rev.Biophys. 17:1 (1984). If there is a second pyrimidine adjacent to apsoralen-pyrimidine monoadduct and on the opposite strand, absorption ofa second photon can lead to formation of a diadduct which functions asan interstrand crosslink. S. T. Isaacs et al., Biochemistry 16:1058(1977); S. T. Isaacs et al., Trends in Photobiology (Plenum) pp. 279-294(1982); J. Tessman et al., Biochem. 24:1669 (1985); Hearst et al., U.S.Pat. Nos. 4,124,598, 4,169,204, and 4,196,281, hereby incorporated byreference.

The covalently bonded psoralens act as inhibitors of DNA replication andthus have the potential to stop the replication process. Due to this DNAbinding capability, psoralens are of particular interest in relation tosolving the problems inherent in creating and maintaining a pathogenblood supply. Some known psoralens have been shown to inactivate virusesin some blood products. H. J. Alter et al., The Lancet (ii:1446) (1988);L. Lin et al., Blood 74:517 (1989) (decontaminating plateletconcentrates); G. P. Wiesehahn et al., U.S. Pat. Nos. 4,727,027 and4,748,120, hereby incorporated by reference, describe the use of acombination of 8-methoxypsoralen (8-MOP) and irradiation. P. Morel etal., Blood Cells 18:27 (1992) show that 300 gg/mL of 8-MOP together withten hours of irradiation with ultraviolet light can effectivelyinactivate viruses in human serum. Similar studies using 8-MOP andaminomethyltrimethyl psoralen (AMT) have been reported by otherinvestigators. Dodd R. Y., et al., Transfusion 31:483-490 (1991);Margolis-Nunno, H., et al., Thromb Haemostas 65:1162 (Abstract)(1991).Indeed, the photoinactivation of a broad spectrum of microorganisms hasbeen established, including HBV, HCV, and HIV, under conditionsdifferent from those used in the present invention and using previouslyknown psoralen derivatives. [Hanson, C. V., Blood Cells: 18:7-24 (1992);Alter, H. J., et al., The Lancet ii:1446 (1988); Margolis-Nunno, H. etal., Thromb Haemostas 65:1162 (Abstract) (1991).]

Psoralen photoinactivation is only feasible if the ability of thepsoralen to inactivate viruses is sufficient to ensure a safety marginin which complete inactivation will occur. On the other hand, thepsoralen must not be such that it will cause damage to blood products.The methods just described, when applied using known psoralens, requirethe use of difficult and expensive procedures to avoid causing damage toblood products. For example, some compounds and protocols havenecessitated the removal of molecular oxygen from the reaction beforeexposure to light, to prevent damage to blood products from oxygenradicals produced during irradiation. See L. Lin et al., Blood 74:517(1989); U.S. Pat. No. 4,727,027, to Wiesehahn. This is a costly and timeconsuming procedure.

Finally, some commonly known compounds used in photochemicaldecontamination (PCD) exhibit undesirable mutagenicity which appears toincrease with increased ability to kill virus. In other words, the moreeffective the known compounds are at inactivating viruses, the moreinjurious the compounds are to the recipient, and thus, the less usefulthey are at any point in an inactivation system of products for in vivouse.

A new psoralen compound is needed which displays improved ability toinactivate bacteria and low mutagenicity, without causing significantdamage to blood products for which it is used, and without the need toremove oxygen, thereby ensuring safe and complete inactivation ofbacteria in blood decontamination methods.

SUMMARY OF THE INVENTION

The present invention provides methods of using new and known compoundsto inactivate bacteria in health related products to be used in vivo andin vitro, and particularly, in blood products and blood products insynthetic media.

The present invention contemplates a method of inactivating bacteria inbiological compositions, comprising, in the following order: a)providing, in any order, i) a compound from the group comprising4'-primaryamino-substituted psoralens and 5'-primaryamino-substitutedpsoralens; ii) photoactivating means for photoactivating said compounds;and iii) a biological composition suspected of being contaminated withbacteria; b) adding said compound to said biological composition; and c)photoactivating said compound, so as to inactivate said bacteria. Thebacteria may comprise a gram negative bacteria or a gram positivebacteria. The biological composition may comprise platelets or plasma.

The present invention contemplates that the photoactivating meanscomprises a photoactivation device capable of emitting a given intensityof a spectrum of electromagnetic radiation comprising wavelengthsbetween 180 nm and 400 nm. The intensity is preferably between 1 and 30mW/cm². In one embodiment, the biological composition is exposed to saidintensity for between 1 second and thirty minutes. Additionally, thespectrum of electromagnetic radiation comprises wavelengths between 320nm and 380 nm.

The present invention contemplates an embodiment of the presentinvention wherein the biological composition is in a synthetic media. Inanother embodiment, the compound is added to said biological compositionto a final concentration of between 1 and 250 μM, preferably, to a finalconcentration of between 10 and 150 μM. It is contemplated that step cmay be performed without limiting the concentration of molecular oxygen.

In one embodiment, the 4'-primaryamino-substituted psoralen comprises:a) a substituent R₁ on the 4' carbon atom, selected from the groupcomprising: --(CH₂)_(u) --NH₂, --(CH₂)_(w) --R₂ --(CH₂)_(z) --NH₂,--(CH₂)_(w) --R₂ --(CH₂)_(x) --R₃ --(CH₂)_(z) --NH₂, and --(CH₂)_(w)--R₂ --(CH₂)_(x) --R₃ --(CH₂)_(y) --R₄ --(CH₂)_(z) --NH₂ ; wherein R₂,R₃, and R₄ are independently selected from the group comprising O andNH, in which u is a whole number from 1 to 10, w is a whole number from1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to5, and z is a whole number from 2 to 6; and b) substituents R₅, R₆, andR₇ on the 4, 5', and 8 carbon atoms respectively, independently selectedfrom the group comprising H and (CH₂)_(v) CH₃, where v is a whole numberfrom 0 to 5; or a salt thereof.

In an alternative embodiment, said 5'-primaryamino-substituted psoralencomprises: a) a substituent R₁ on the 5' carbon atom, selected from thegroup comprising: --(CH₂)_(u) --NH₂, --(CH₂)_(w) --R₂ --(CH₂)_(z) --NH₂,--(CH₂)_(w) --R₂ --(CH₂)_(x) --R₃ --(CH₂)_(z) --NH₂, and --(CH₂)_(w)--R₂ --(CH₂)_(x) --R₃ --(CH₂)_(y) --R₄ --(CH₂)_(z) --NH₂ ; wherein R₂,R₃, and R₄ are independently selected from the group comprising O andNH, and in which u is a whole number from 1 to 10, w is a whole numberfrom 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2to 5, and z is a whole number from 2 to 6; and, b) substituents R₅, R₆,and R₇ on the 4, 4', and 8 carbon atoms respectively, independentlyselected from the group comprising H and (CH₂)_(v) CH₃, where v is awhole number from 0 to 5, where when R₁ is selected from the groupcomprising --(CH₂)_(u) --NH₂, R₇ is (CH₂)_(v) CH₃, and where when R₅,R₆, and R₇ are (CH₂)_(v) CH₃, u is a whole number from 3 to 10; or asalt thereof. The present invention further contemplates an embodimentwherein at least two of said compounds are present. The compound may bein a solution before step c, and said solution may comprise water,saline, or a synthetic media. Additionally, the present inventioncontemplates an embodiment wherein said synthetic media furthercomprises sodium acetate, potassium chloride, sodium chloride, sodiumcitrate, sodium phosphate and magnesium chloride, and may also compriseat least one constituent selected from the group consisting of mannitoland glucose.

The present invention further contemplates an embodiment wherein beforestep (b) said solution is contained in a first blood bag and saidplatelet preparation is contained in a second blood bag. Further, saidsolution may be added to said platelet preparation in step (b) byexpressing said solution from said first blood bag into said secondblood bag via a sterile connection. The present invention contemplatesthat the compound used in this method may comprise4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen.

It is contemplated further that said platelet preparation may beadministered by intravenous infusion to a mammal. It is alsocontemplated that said compound be in a dry formulation before step c.

In yet another embodiment, the present invention contemplates a methodof inactivating bacteria in biological compositions, comprising, in thefollowing order: a) providing, in any order, i)4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen; ii) photoactivatingmeans for photoactivating said4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen; and iii) a biologicalcomposition suspected of being contaminated with bacteria; b) addingsaid 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen to said biologicalcomposition; and c) photoactivating said4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen, so as to inactivatesaid bacteria. The bacteria decontaminated by this method may comprise agram negative bacteria or a gram positive bacteria. It is alsocontemplated that the biological composition comprise platelets orplasma. The present invention also contemplates that the photoactivatingmeans comprises a photoactivation device capable of emitting a givenintensity of a spectrum of electromagnetic radiation comprisingwavelengths between 180 nm and 400 nm, preferrably with an intensity ofbetween 1 and 30 mW/cm². In one embodiment, the biological compositionis exposed to said intensity for between 1 second and thirty minutes.Additionally, the spectrum of electromagnetic radiation compriseswavelengths between 320 nm and 380 nm.

The present invention contemplates an embodiment of the presentinvention wherein the biological composition is in a synthetic media. Inanother embodiment, the compound is added to said biological compositionto a final concentration of between 0.1 and 250 μM. It is contemplatedthat step c may be performed without limiting the concentration ofmolecular oxygen. The compound may be in a solution before step c, andsaid solution may comprise water, saline, or a synthetic media.Additionally, the present invention contemplates an embodiment whereinsaid synthetic media further comprises sodium acetate, potassiumchloride, sodium chloride, sodium citrate, sodium phosphate andmagnesium chloride, and may also comprise at least one constituentselected from the group consisting of mannitol and glucose.

The present invention further contemplates an embodiment wherein beforestep (b) said solution is contained in a first blood bag and saidplatelet preparation is contained in a second blood bag. Further, saidsolution may be added to said platelet preparation in step (b) byexpressing said solution from said first blood bag into said secondblood bag via a sterile connection. The present invention contemplatesthat the compound used in this method may comprise4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen.

It is contemplated further that said platelet preparation may beadministered by intravenous infusion to a mammal. It is alsocontemplated that said compound be in a dry formulation before step c.

DESCRIPTION OF THE DRAWINGS

FIG. 1 details the compound synthesis scheme of two isomers ofbromomethyl-trialkylpsoralen.

FIG. 2 details the compound synthesis scheme of several 4'-primary aminosubstituted psoralens.

FIG. 3 details the compound synthesis scheme of several 4'-primaryaminosubstituted psoralens.

FIG. 4 details the compound synthesis scheme of several psoralens havinga terminal amine linked to the psoralen at the 4'-position by an alkylchain.

FIG. 5 details the compound synthesis scheme of several5'-primaryamino-substituted psoralens.

FIG. 6 is a perspective view of one embodiment of the device of thepresent invention.

FIG. 7 is a cross-sectional view of the device shown in FIG. 6 along thelines of 2--2.

FIG. 8 is a cross-sectional view of the device shown in FIG. 6 along thelines of 3--3.

FIG. 9 is a cross-sectional view of the device shown in FIG. 6 along thelines of 4--4.

FIG. 10A is a diagram of synthesis pathways and chemical structures ofcompounds 8, 13, and 14 of the present invention.

FIG. 10B is a diagram of synthesis pathways and chemical structures ofcompounds 2, 4, and 7 of the present invention.

FIG. 10C is a diagram of synthesis pathways and chemical structures ofcompounds 1, 5, 6, 9, and 10 of the present invention.

FIG. 10D is a diagram of synthesis pathways and chemical structures ofcompounds 12 and 15 of the present invention.

FIG. 10E is a diagram of a synthesis pathway and the chemical structureof compound 3 of the present invention.

FIG. 10F is a diagram of synthesis pathways and the chemical structureof compounds 16 and 17 of the present invention.

FIG. 11 shows the impact of concentration on the log kill of R17 whenCompounds 1-3 of the present invention are photoactivated.

FIG. 12 shows the impact of concentration on the log kill of R17 whenCompounds 3-6 of the present invention are photoactivated.

FIG. 13 shows the impact of concentration on the log kill of R17 whenCompounds 2 and 6 of the present invention are photoactivated.

FIG. 14 shows the impact of concentration on the log kill of R17 whenCompounds 6 and 18 of the present invention are photoactivated.

FIG. 15 shows the impact of concentration on the log kill of R17 whenCompound 16 of the present invention is photoactivated.

FIG. 16 shows the impact of varying Joules/cm² (Watt second/cm²) ofirradiation on the log titer of R17 for Compound 6 of the presentinvention.

FIG. 17 shows the impact of varying Joule/cm² of irradiation on the logtiter of R17 for Compounds 7, 9 and 10 of the present invention.

FIG. 18 shows the impact of varying Joules/cm² of irradiation on the logtiter of R17 for Compounds 7 and 12 of the present invention.

FIG. 19 shows the impact of varying Joules/cm² of irradiation on the logtiter of R17 for Compound 15 of the present invention.

FIG. 20 shows the impact of varying Joules/cm² of irradiation on the logtiter of R17 for Compound 17 of the present invention.

FIG. 21 shows the impact of varying Joules/cm² of irradiation on the logtiter of R17 for Compounds 6 and 17 of the present invention.

FIG. 22 shows the impact of varying Joules/cm² of irradiation on the logtiter of R17 for Compounds 6 and 15 of the present invention.

FIG. 23 shows the effect of varying the concentration of Compounds 2 and6 of the present invention, in plasma.

FIG. 24 shows the effect of varying the concentration of Compounds 2 and6 of the present invention, in synthetic medium.

FIG. 25A schematically shows the standard blood product separationapproach used presently in blood banks.

FIG. 25B schematically shows an embodiment of the present inventionwhereby synthetic media is introduced to platelet concentrate preparedas in FIG. 25A.

FIG. 25C schematically shows one embodiment of the decontaminationapproach of the present invention applied specifically to plateletconcentrate diluted with synthetic media as in FIG. 25B.

FIG. 26A is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 2 at 100 μM (PCD) by their effects onplatelet function as measured by platelet count. "n" represents thenumber of experiments represented by the data point.

FIG. 26B is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 2 at 100 μM (PCD) by their effects onplatelet function as measured by platelet aggregation. "n" representsthe number of experiments represented by the data point.

FIG. 26C, is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 2 at 100 μM (PCD) by their effects onplatelet function as measured by GMP-140 expression. "n" represents thenumber of experiments represented by the data point.

FIG. 26D is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 2 at 100 μM (PCD) by their effects onplatelet function as measured by pH. "n" represents the number ofexperiments represented by the data point.

FIG. 27A is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 6 at 100 μM (PCD) by their effects onplatelet function as measured by platelet count. "n" represents thenumber of experiments represented by the data point.

FIG. 27B is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 6 at 100 μM (PCD) by their effects onplatelet function as measured by platelet aggregation. "n" representsthe number of experiments represented by the data point.

FIG. 27C is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 6 at 100 μM (PCD) by their effects onplatelet function as measured by GMP-140 expression. "n" represents thenumber of experiments represented by the data point.

FIG. 27D is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 6 at 100 μM (PCD) by their effects onplatelet function as measured by pH. "n" represents the number ofexperiments represented by the data point.

FIG. 28A is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 17 at 100 μM (PCD) by their effects onplatelet function as measured by platelet count. "n" represents thenumber of experiments represented by the data point.

FIG. 28B is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 17 at 100 μM (PCD) by their effects onplatelet function as measured by platelet aggregation. "n" representsthe number of experiments represented by the data point.

FIG. 28C is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 17 at 100 μM (PCD) by their effects onplatelet function as measured by GMP-140 expression. "n" represents thenumber of experiments represented by the data point.

FIG. 28D is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 17 at 100 μM (PCD) by their effects onplatelet function as measured by pH. "n" represents the number ofexperiments represented by the data point.

FIG. 29A is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 18 at 100 μM (PCD) by their effects onplatelet function as measured by platelet count. "n" represents thenumber of experiments represented by the data point.

FIG. 29B is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 18 at 100 μM (PCD) by their effects onplatelet function as measured by platelet aggregation. "n" representsthe number of experiments represented by the data point.

FIG. 29C is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 18 at 100 μM (PCD) by their effects onplatelet function as measured by GMP-140 expression. "n" represents thenumber of experiments represented by the data point.

FIG. 29D is a graph comparing a day one control (C1), a control storedfor 5 days (D5), ultraviolet light alone (uv) and treatment withultraviolet light and Compound 18 at 100 μM (PCD) by their effects onplatelet function as measured by pH. "n" represents the number ofexperiments represented by the data point.

DESCRIPTION OF THE INVENTION

The present invention provides methods of inactivating bacteria inhealth related products to be used in vivo and in vitro, and inparticular, blood products, which do not significantly effect bloodproduct function or exhibit mutagenicity.

The inactivation methods of the present invention provide methods ofinactivating pathogens, and in particular, bacteria, in blood productsprior to use in vitro or in vivo. In contrast with previous approaches,the method requires only short irradiation times and there is no need tolimit the concentration of molecular oxygen.

The description of the invention is divided into the following sections:I) Photoactivation Devices, II) Compound Synthesis, III) Binding ofCompounds to Nucleic Acid, IV) Inactivation of Contaminants, and V)Preservation of Biochemical Properties of Material Treated.

I. PHOTOACTIVATION DEVICES

The present invention contemplates devices and methods forphotoactivation and specifically, for photoactivation of photoreactivenucleic acid binding compounds. The present invention contemplatesdevices having an inexpensive source of electromagnetic radiation thatis integrated into a unit. In general, the present inventioncontemplates a photoactivation device for treating photoreactivecompounds, comprising: a) means for providing appropriate wavelengths ofelectromagnetic radiation to cause photoactivation of at least onephotoreactive compound; b) means for supporting a plurality of samplesin a fixed relationship with the radiation providing means duringphotoactivation; and c) means for maintaining the temperature of thesamples within a desired temperature range during photoactivation. Thepresent invention also contemplates methods, comprising: a) supporting aplurality of sample containers, containing one or more photoreactivecompounds, in a fixed relationship with a fluorescent source ofelectromagnetic radiation; b) irradiating the plurality of samplecontainers simultaneously with electromagnetic radiation to causephotoactivation of at least one photoreactive compound; and c)maintaining the temperature of the sample within a desired temperaturerange during photoactivation.

The major features of one embodiment of the device of the presentinvention involve: A) an inexpensive source of ultraviolet radiation ina fixed relationship with the means for supporting the samplecontainers, B) rapid photoactivation, C) large sample processing, D)temperature control of the irradiated samples, and E) inherent safety.

A. Electromagnetic Radiation Source

Many sources of ultraviolet radiation can be successfully used indecontamination protocols with psoralens. For example, some groups haveirradiated sample from above and below by General Electric typeF20T12-BLB fluorescent UVA bulbs with an electric fan blowing gentlyacross the lights to cool the area. Alter, H. J., et al., The Lancet,24:1446 (1988). Another group used Type A405-TLGW/05 long wavelengthultraviolet lamp manufactured by P. W. Allen Co., London placed abovethe virus samples in direct contact with the covers of petri dishescontaining the samples, and was run at room temperature. The totalintensity delivered to the samples under these conditions was 1.3×10¹⁵photons/second cm² (or 0.7 mW/cm² or 0.0007 J/cm² sec) in the petridish. Hearst, J. E., and Thiry, L., Nucleic Acids Research, 4:1339(1977). However, without intending to be limited to any type ofphotoactivation device, the present invention contemplates severalpreferred arrangements for the photoactivation device, as follows.

A preferred photoactivation device of the present invention has aninexpensive source of ultraviolet radiation in a fixed relationship withthe means for supporting the sample vessels. Ultraviolet radiation is aform of energy that occupies a portion of the electromagnetic radiationspectrum (the electromagnetic radiation spectrum ranges from cosmic raysto radio waves). Ultraviolet radiation can come from many natural andartificial sources. Depending on the source of ultraviolet radiation, itmay be accompanied by other (non-ultraviolet) types of electromagneticradiation (e.g. visible light).

Particular types of ultraviolet radiation are herein described in termsof wavelength. Wavelength is herein described in terms of nanometers("nm"; 10⁻⁹ meters). For purposes herein, ultraviolet radiation extendsfrom approximately 180 nm to 400 nm. When a radiation source, by virtueof filters or other means, does not allow radiation below a particularwavelength (e.g. 320 nm), it is said to have a low end "cutoff" at thatwavelength (e.g. "a wavelength cutoff at 300 nanometers"). Similarly,when a radiation source allows only radiation below a particularwavelength (e.g. 360 nm), it is said to have a high end "cutoff" at thatwavelength (e.g. "a wavelength cutoff at 360 nanometers").

For any photochemical reaction it is desired to eliminate or leastminimize any deleterious side reactions. Some of these side reactionscan be caused by the excitation of endogenous chromophores that may bepresent during the photoactivation procedure. In a system where onlynucleic acid and psoralen are present, the endogenous chromophores arethe nucleic acid bases themselves. Restricting the photoactivationprocess to wavelengths greater than 320 nm minimizes direct nucleic aciddamage since there is very little absorption by nucleic acids above 313nm.

In human serum or plasma, for example, the nucleic acid is typicallypresent together with additional biological constituents. If thebiological fluid is just protein, the 320 nm cutoff will be adequate forminimizing side reactions (aromatic amino acids do not absorb above 320nm). If the biological fluid includes other analytes, there may beconstituents that are sensitive to particular wavelengths of light. Inview of the presence of these endogenous constituents, it is intendedthat the device of the present invention be designed to allow forirradiation within a small range of specific and desirable wavelengths,and thus avoid damage blood components. The preferred range of desirablewavelengths is between 320 and 350 nm.

Some selectivity can be achieved by choice of commercial irradiationsources. For example, while typical fluorescent tubes emit wavelengthsranging from 300 nm to above 400 nm (with a broad peak centered around360 nm), BLB type fluorescent lamps are designed to remove wavelengthsabove 400 nm. This, however, only provides an upper end cutoff.

In a preferred embodiment, the device of the present invention comprisesan additional filtering means. In one embodiment, the filtering meanscomprises a glass cut-off filter, such as a piece of Cobalt glass. Inone embodiment, the filtering means is BK-7 glass, available from ShottGlass Technologies, Inc. Duryea, Pa. In another embodiment, thefiltering means comprises a liquid filter solution that transmits only aspecific region of the electromagnetic spectrum, such as an aqueoussolution of Co(No₃)₂. This salt solution yields a transmission window of320-400 nm. In a preferred embodiment, the aqueous solution of Co(No₃)₂is used in combination with NiSO₄ to remove the 365 nm component of theemission spectrum of the fluorescent or arc source employed. The Co--Nisolution preserves its initial transmission remarkably well even aftertens of hours of exposure to the direct light of high energy sources.

It is not intended that the present invention be limited by theparticular filter employed. Several inorganic salts and glasses satisfythe necessary requirements. For example. cupric sulfate is a most usefulgeneral filter for removing the infra-red, when only the ultraviolet isto be isolated. Its stability in intense sources is quite good. Othersalts are known to one skilled in the art. Aperture or reflector lampsmay also be used to achieve specific wavelengths and intensities.

When ultraviolet radiation is herein described in terms of irradiation,it is expressed in terms of intensity flux (milliwatts per squarecentimeter or "mW cm-2" or J/cm² sec). "Output" is herein defined toencompass both the emission of radiation (yes or no; on or off) as wellas the level of irradiation. In a preferred embodiment, intensity ismonitored at 4 locations: 2 for each side of the plane of irradiation.

A preferred source of ultraviolet radiation is a fluorescent source.Fluorescence is a special case of luminescence. Luminescence involvesthe absorption of electromagnetic radiation by a substance and theconversion of the energy into radiation of a different wavelength. Withfluorescence, the substance that is excited by the electromagneticradiation returns to its ground state by emitting a quantum ofelectromagnetic radiation. While fluorescent sources have heretoforebeen thought to be of too low intensity to be useful forphotoactivation, in one embodiment the present invention employsfluorescent sources to achieve results thus far achievable on onlyexpensive equipment.

As used here, fixed relationship is defined as comprising a fixeddistance and geometry between the sample and the light source during thesample irradiation. Distance relates to the distance between the sourceand the sample as it is supported. It is known that light intensity froma point source is inversely related to the square of the distance fromthe point source. Thus, small changes in the distance from the sourcecan have a drastic impact on intensity. Since changes in intensity canimpact photoactivation results, changes in distance are avoided in thedevices of the present invention. This provides reproducibility andrepeatability.

Geometry relates to the positioning of the light source. For example, itcan be imagined that light sources could be placed around the sampleholder in many ways (on the sides, on the bottom, in a circle, etc.).The geometry used in a preferred embodiment of the present inventionallows for uniform light exposure of appropriate intensity for rapidphotoactivation. The geometry of a preferred device of the presentinvention involves multiple sources of linear lamps as opposed to singlepoint sources. In addition, there are several reflective surfaces andseveral absorptive surfaces. Because of this complicated geometry,changes in the location or number of the lamps relative to the positionof the samples to be irradiated are to be avoided in that such changeswill result in intensity changes.

B. Rapid Photoactivation

The light source of the preferred embodiment of the present inventionallows for rapid photoactivation. The intensity characteristics of theirradiation device have been selected to be convenient with theanticipation that many sets of multiple samples may need to beprocessed. With this anticipation, a fifteen minute exposure time orless is a practical goal.

In designing the devices of the present invention, relative position ofthe elements of the preferred device have been optimized to allow forapproximately fifteen minutes of irradiation time, so that, whenmeasured for the wavelengths between 320 and 350 nanometers, anintensity flux greater than approximately 1 mW cm-² (0.001 J/cm² sec.)is provided to the sample vessels.

C. Processing of Large Numbers of Samples

As noted, another important feature of the photoactivation devices ofthe present invention is that they provide for the processing of largenumbers of samples. In this regard, one element of the devices of thepresent invention is a means for supporting a plurality of blood bags.In the preferred embodiment of the present invention the supportingmeans comprises a blood bag support placed between two banks of lights.By accepting commonly used commercially available bags, the device ofthe present invention allows for convenient processing of large numbersof samples.

D. Temperature Control

As noted, one of the important features of the photoactivation devicesof the present invention is temperature control. Temperature control isimportant because the temperature of the sample in the sample at thetime of exposure to light can dramatically impact the results. Forexample, conditions that promote secondary structure in nucleic acidsalso enhance the affinity constants of many psoralen derivatives fornucleic acids. Hyde and Hearst, Biochemistry, 17, 1251 (1978). Theseconditions are a mix of both solvent composition and temperature. Withsingle stranded 5S ribosomal RNA, irradiation at low temperaturesenhances the covalent addition of HMT to 5S rRNA by two fold at 4° C.compared to 20° C. Thompson et al., J. Mol. Biol. 147:417 (1981). Evenfurther temperature induced enhancements of psoralen binding have beenreported with synthetic polynucleotides. Thompson et al., Biochemistry21:1363 (1982).

E. Inherent Safety

Ultraviolet radiation can cause severe burns. Depending on the nature ofthe exposure, it may also be carcinogenic. The light source of apreferred embodiment of the present invention is shielded from the user.This is in contrast to the commercial hand-held ultraviolet sources aswell as the large, high intensity sources. In a preferred embodiment,the irradiation source is contained within a housing made of materialthat obstructs the transmission of radiant energy (i.e. an opaquehousing). No irradiation is allowed to pass to the user. This allows forinherent safety for the user.

II. COMPOUND SYNTHESIS

A. Photoactivation Compounds in General

"Photoactivation compounds" (or "photoreactive compounds") defines afamily of compounds that undergo chemical change in response toelectromagnetic radiation. Table 1 is a partial list of photoactivationcompounds.

Table 1.

Photoactivation Compounds

Actinomycins

Anthracyclinones

Anthramycin

Benzodipyrones

Fluorenes and fluorenones

Furocoumarins

Mitomycin

Monostral Fast Blue

Norphillin A

Many organic dyes not specifically listed

Phenanthridines

Phenazathionium Salts

Phenazines

Phenothiazines

Phenylazides

Quinolines

Thiaxanthenones

The preferred species of photoreactive compounds described herein iscommonly referred to as the furocoumarins. In particular, the presentinvention contemplates those compounds described as psoralens:[7H-furo(3,2-g)-(1)-benzopyran-7-one, or β-lactone of6-hydroxy-5-benzofuranacrylic acid], which are linear: ##STR1## and inwhich the two oxygen residues appended to the central aromatic moietyhave a 1, 3 orientation, and further in which the furan ring moiety islinked to the 6 position of the two ring coumarin system. Psoralenderivatives are derived from substitution of the linear furocoumarin atthe 3, 4, 5, 8, 4', or 5' positions.

8-Methoxypsoralen (known in the literature under various named, e.g.,xanthotoxin, methoxsalen, 8-MOP) is a naturally occurring psoralen withrelatively low photoactivated binding to nucleic acids and lowmutagenicity in the Ames assay, which is described in the followingexperimental section. 4'-Aminomethyl-4,5',8-trimethylpsoralen (AMT) isone of most reactive nucleic acid binding psoralen derivatives,providing up to 1 AMT adduct per 3.5 DNA base pairs. S. T. Isaacs, G.Wiesehahn and L. M. Hallick, NCI Monograph 66: 21 (1984). However, AMTalso exhibits significant levels of mutagenicity. A new group ofpsoralens was desired which would have the best characteristics of both8-MOP and AMT: low mutagenicity and high nucleic acid binding affinity,to ensure safe and thorough inactivation of pathogens. The compounds ofthe present invention were designed to be such compounds.

"4'-primaryamino-substituted psoralens" are defined as psoralencompounds which have an NH₂ group linked to the 4'-position of thepsoralen by a hydrocarbon chain having a total length of 2 to 20carbons, where 0 to 6 of those carbons are independently replaced by NHor O, and each point of replacement is separated from each other pointof replacement by at least two carbons, and is separated from thepsoralen by at least one carbon 4'-primaryamino-substituted psoralensmay have additional substitutions on the 4, 5', and 8 positions of thepsoralen, said substitutions include, but are not limited to, thefollowing groups: H and (CH₂)_(n) CH₃, where n=0-6.

"5'-primaryamino-substituted psoralens" are defined as psoralencompounds which have an NH₂ group linked to the 5'-position of thepsoralen by a hydrocarbon chain having a total length of 1 to 20carbons, where 0 to 6 of those carbons are independently replaced by NHor O, and each point of replacement is separated from each other pointof replacement by at least two carbons, and is separated from thepsoralen by at least one carbon. 5'-primaryamino-substituted psoralensmay have additional substitutions on the 4, 4', and 8 positions of thepsoralen, said subsitutions include, but are not limited to, thefollowing groups: H and (CH₂)_(n) CH₃, where n=0-6.

B. Synthesis of the Psoralens

The present invention contemplates synthesis methods for the novelcompounds of the present invention, as well as new synthesis methods forknown intermediates. Specifically, the novel compounds are mono, di ortrialkylated 4'- or 5'-primaryamino-substituted psoralens. Severalexamples of the schemes discussed in this section are shown in FIGS.10A-10F. For ease of reference, TABLE 2 sets forth the nomenclature usedfor the psoralen derivatives discussed herein. The structures ofcompounds 1-18 are also pictured in FIGS. 10A-10F. Note that thissection (entitled "B. Synthesis of the Psoralens") the roman numeralsused to identify compounds correlate with Schematics 1-6 only, and donot correlate with the compound numbers listed in Table 2 or FIGS.10A-10F.

                  TABLE 2                                                         ______________________________________                                        #       COMPOUND                                                              ______________________________________                                        1       4'-(4-amino-2-aza)butyl-4,5',8-trimethylpsoralen                      2       4'-(4-amino-2-oxa)butyl4,5',8-trimethylpsoralen                       3       4'-(2-aminoethyl)-4,5',8-trimethylpsoralen                            4       4'-(5-amino-2-oxa)pentyl-4,5',8-trimethylpsoralen                     5       4'-(5-amino-2-aza)pentyl-4,5',8-trimethylpsoralen                     6       4'-(6-amino-2-aza)hexyl-4,5',8-trimethylpsoralen                      7       4'-(7-amino-2,5-oxa)heptyl-4,5',8-                                            trimethylpsoralen                                                     8       4'-(12-amino-8-aza-2,5-dioxa)dodecyl-4,5',8-                                  trimethylpsoralen                                                     9       4'-(13-amino-2-aza-6,11-dioxa)tridecyl-4,5',8-                                trimethylpsoralen                                                     10      4'-(7-amino-2-aza)heptyl-4,5',8-trimethylpsoralen                     11      4'-(7-amino-2-aza-5-oxa)heptyl-4,5',8-                                        trimethylpsoralen                                                     12      4'-(9-amino-2,6-diaza)nonyl-4,5',8-                                           trimethylpsoralen                                                     13      4'-(8-amino-5-aza-2-oxa)octyl-4,5',8-                                         trimethylpsoralen                                                     14      4'-(9-amino-5-aza-2-oxa)nonyl-4,5',8-                                         trimethylpsoralen                                                     15      4'-(14-amino-2,6,11-triaza)tetradecyl-4,5',8-                                 trimethylpsoralen                                                     16      5'-(4-amino-2-aza)butyl-4,4',8-trimethylpsoralen                      17      5'-(6-amino-2-aza)hexyl-4,4',8-trimethylpsoralen                      18      5'-(4-amino-2-oxa)butyl-4,4',8-trimethylpsoralen                      ______________________________________                                    

It is most logical to first describe the synthesis of intermediatesuseful in synthesizing many of the compounds of the present invention.While the invention is not limited to4,5',8-trimethyl-4'-primaryamino-substituted psoralens or4,4',8-trimethyl-5'-primaryamino-substituted psoralens, some importantintermediates include tri- and tetramethyl psoralens,4'-halomethyl-4,5',8-trimethylpsoralens and5'-halomethyl-4,4',8-trimethylpsoralens. The preparation of thesecritical intermediates presents difficult challenges.

Synthesis of Intermediates

Previous syntheses of 4'-chloromethyl-4,5',8-trimethylpsoralen (4'-CMT)and 4'-bromomethyl-4,5',8-trimethylpsoralen (4'-BrMT) start from4,5',8-trimethylpsoralen (5'-TMP) which is commercially available(Aldrich Chemical Co., Milwaukee, Wis.) or can be prepared in four stepsas described below for other alkylated psoralens. 5'-TMP is converted to4'-CMT using a large excess (20-50 equivalents) of highly carcinogenic,and volatile chloromethyl methyl ether. Halomethylation of the4,5',8-trialkylpsoralens with chloromethyl methyl ether or bromomethylmethyl ether is described in U.S. Pat. No. 4,124,598, to Hearst. Thebromo compound, 4'-BrMT, is likewise prepared using bromomethyl methylether which is somewhat less volatile. Yields of only 30-60% of thedesired intermediate are obtained. The5'-chloromethyl-4,4',8-trimethylpsoralen (5'-CMT) and5'-bromomethyl-4,4',8-trimethylpsoralen (5'-BrMT) are preparedsimilarly, using the isomeric starting compound,4,4',8-trimethylpsoralen (4'-TMP) [U.S. Pat. No. 4,294,822, to Kaufman;McLeod, et al., "Synthesis of Benzofuranoid Systems. I. Furocoumarins,Benzofurans and Dibenzofurans," Tetrahedron Letters 237 (1972)]. Some hefigures referred to in the synthesis descussion that follows containroman numerals used for labeling structures that embody more than onecompound. This numbering system is distinct from, and not to be confusedwith, the numbering system of Table 2, above, which was used to identifyseveral specific compounds.

Described herein is a much improved procedure which allows for thesynthesis of either isomer of the bromomethyl-trialkylpsoralens from thesame psoralen precursor by careful control of reaction conditions. SeeFIG. 1. In FIG. 1, A₁ and A₂ are independently selected from the groupcomprising H and an alkyl chain having 1-6 carbon atoms. Reaction of the4,8-dialkyl-7-hydroxycoumarin with 2-chloro-3-butanone under typicalbasic conditions, provides4,8-dialkyl-7-(1-methyl-2-oxo)propyloxycoumarin (I). This material iscyclized by heating in aqueous NaOH to provide4,8-dialkyl-4',5'-dimethylpsoralen (II). Treatment of thetetrasubstituted psoralen and N-bromosuccinimide (NBS) in a solvent atroom temperature up to 150° C. leads to bromination at the 4'- or 5'-position, depending upon the conditions used. A catalyst such asdibenzoyl peroxide may be added, but is not necessary. If the solventused is carbon tetrachloride at reflux,4,8-dialkyl-5'-bromomethyl-4'-methylpsoralen (IV) is obtained in yieldsof 50% or greater. If methylene chloride is used at room temperature,only 4,8-dialkyl-4'-bromomethyl-5'-methylpsoralen (III) is obtainedin >80% yield. Benzylic bromination in other solvents can also be done,generating one of the isomeric products alone or in a mixture. Thesesolvents include, but are not limited to 1,2-dichloroethane, chloroform,bromotrichloromethane and benzene.

General Scheme of Synthesis of 4'-Substituted Psoralens

Turning now to the synthesis of a subclass of the linear psoralens,4,5',8-trialkylpsoralens can be made as follows. The4,8-dialkylcoumarins are prepared from 2-alkylresorcinols and a3-oxoalkanoate ester by the Pechmann reaction (Organic Reactions VolVII, Chap 1, ed. Adams et al, Wiley, N.Y., (1953)). The hydroxy group istreated with an allylating reagent, CH₂ ═CHX--CH(R)--Y, where X is ahalide or hydrogen, Y is a halide or sulfonate, and R is H or (CH₂)_(v)CH₃, where v is a whole number from 0 to 4. Claisen rearrangement of theresultant allyl ether gives 4,8-dialkyl-6-allyl-7-hydroxycoumarin. Thecoumarins are converted to the 4,5',8-trialkylpsoralens using proceduressimilar to one of several previously described procedures (i.e., see,Bender et al, J. Org. Chem. 44:2176 (1979); Kaufman, U.S. Pat. Nos.4,235,781 and 4,216,154, hereby incorporated by reference).4,5',8-Trimethylpsoralen is a natural product and is commerciallyavailable (Aldrich Chemical Co., Milwaukee, Wis.).

General Scheme of Synthesis of 5'-Substituted Psoralens

The 4,4',8-trialkylpsoralens can be prepared in two steps also startingfrom the 4,8-dialkyl-7-hydroxycoumarins discussed above. The coumarin istreated with an alpha-chloro ketone under basic conditions to give the4,8-dialkyl-7-(2-oxoalkoxy)coumarin. Cyclization of this intermadiate tothe 4,4',8-trialkylcoumarin occurs by heating in aqueous base.

Longer chain 4'-(ω-haloalkyl)trialkylpsoralens (herein referred to aslonger chain 4'-HATP) where the alkyl groups are selected from the group(CH₂)₂ to (CH₂)₁₀ can be prepared under Freidel-Crafts conditions asdiscussed elsewhere (Olah and Kuhn, J. Org. Chem., 1964, 29, 2317;Friedel-Crafts and Related Reactions, Vol. II, Part 2, Olah, ed.,Interscience, N.Y., 1964, p 749). While reactions of the halomethyl-intermediates with amines (e.g., Hearst et al., U.S. Pat. No.4,124,598), and alcohols (e.g., Kaufman, U.S. Pat. No. 4,269,852) havebeen described, there are only two original reports on the formation ofextended chain primary amines. They describe the reaction of the4'-chloromethyl-4,5',8-trimethyl psoralen with H₂ N-(CH₂)_(n) --NH₂(where n=2, 4, 6) (Lee, B., et al. "Interaction of Psoralen-DerivatizedOligodeoxyribonucleoside Methylphosphonates with Single-Stranded DNA,"Biochemistry 27:3197 (1988), and with H₂ NCH₂ CH₂ SSCH₂ CH₂ NH₂(Goldenberg, M., et al., "Synthesis and Properties of Novel PsoralenDerivatives," Biochemistry 27:6971 (1988)). The utility of the resultingcompounds for nucleic acid photoreaction has not previously beenreported. The properties of these materials, such as decreasedmutagenicity, are unexpected based on what is known about previouslyprepared compounds, such as AMT.

Several synthesis routes are shown in FIG. 2. Starting from the 4'-HATP(where w is a number from 1-5; A₁, A₂ and A₃ are independently selectedfrom the group comprising H and (CH₂)_(v) CH₃, where v is a number from0 to 5; and where X=Br, Cl or I), reaction with an excess of abis-hydroxy compound, HO--(B)--OH, where B is either: an alkyl chain(e.g., HO--(B)--OH is 1,3-propanediol), a monoether (e.g., diethyleneglycol) or a polyether (e.g., tetraethylene glycol); which is less thanor equal to 18 carbon atoms long, either neat or with a solvent such asacetone at 20°-80° C., and a base for the carbon chains longer thanhalomethyl, gives a (ω-hydroxyalkoxy)alkyl psoralen. The terminalhydroxy group can be transformed to an amino group under a variety ofconditions (for example see Larock, "Comprehensive OrganicTransformations," VCH Publishers, New York, 1989). Particularly, thehydroxy group can be converted to the ester of methanesulfonic acid(structure VI) in the presence of methanesulphonyl chloride (CH₃ SO₃Cl). This can subsequently be converted to the azide in refluxingethanol and the azide reduced to the final amine, structure VII(examples are Compounds 2, 4 and 7). The method described hereinutilizes triphenylphosphine and water in tetrahydrofuran (THF) for thereduction but other methods are contemplated.

A preferred method of preparation of structure VII uses the reaction of4'-HATP with a primary linear alcohol containing a protected amine(e.g., a phthalimido group) at the terminal position in a suitablesolvent such as DMF at 25°-150° C. to give V. The amine is thendeprotected under standard conditions (e.g., hydrazine or aqueous MeNH₂to deprotect a phthalimido group [higher alkyl hydrazines, such asbenzyl hydrazines, are also contemplated]) to give VII.

Conversely, structure VI can be reacted with diamines, H₂ N-(B')-NH₂where B' is an alkyl chain (e.g., 1,4,-butanediamine), a monoether(e.g., 3-oxa-1,5-pentanediamine) or a polyether (e.g.,3,6-dioxa-1,8-octanediamine) to give the final product, compound VIII(examples of compounds in this structure group are Compounds 8, 13 and14). This reaction is carried out with an excess of diamine inacetonitrile at reflux, but other solvents and temperatures are equallypossible.

Some final compounds are desired in which the carbon chain is linked tothe 4'- position of the psoralen ring by an aminoalkyl group[NH(CH₂)_(w) ] rather than by an oxyalkyl group [O(CH₂)_(w) ]. Synthesispathways for these compounds are shown in FIG. 3. When the linkagebetween this nitrogen and the terminating nitrogen contains only CH₂subunits and oxygen but no other nitrogens (structure X) (examples areCompounds 1, 5, 6, 9, 10 and 11), the product can conveniently beprepared from the 4'-HATP and the appropriate diamine of structure IX.This method is also applicable to final products that contain more thantwo nitrogens in the chain (structure XIII) (examples are Compounds 12and 15) starting from polyamines of structure XII (e.g., norspermidineor spermine [commercially available from Aldrich, Milwaukee, Wis.]),however, in this case isomeric structures are also formed inconsiderable amounts. The preferred method for the preparation ofstructure XIII is reductive amination of the psoralen-4'-alkanal (XI)with a polyamine of structure XII and a reducing agent such as sodiumcyanoborohydride. This reductive amination is applicable to thesynthesis of compounds X as well. The carboxaldehydes (structure XI,w=0) have been prepared previously by hydrolysis of the 4'-halomethylcompounds and subsequent oxidation of the resultant 4'-hydroxymethylcompound. (Isaacs et al, J. Labelled Cmpds. Radiopharm., 1982, 19, 345).These compounds can also be conveniently prepared by formylation of the4'-hydrido compounds with a formamide and POCl₃, or with hexamethylenetetraamine in acid. Longer chain alkanals can be prepared from the4'-HATP compounds by conversion of the terminal halo group to analdehyde functionality (for example, Durst, Adv. Org. Chem. 6:285(1969)).

Other final products have a terminal amine linked to the psoralen by analkyl chain. As shown in FIG. 4, these compounds (structures XIV) (anexample is Compound 3) are prepared either 1) by reaction of the 4'-HATPwith potassium phthalimide or azide and subsequent liberation of thedesired amine as before, for example, with hydrazine, or 2) conversionof the 4'-HATP to the cyanide compound, followed by reduction, forexample with NaBH₄ --CF₃ CO₂ H.

The discussion of the conversion of 4,5',8-trialkylpsoralens to4'-aminofunctionalized-4,5',8-trialkylpsoralens applies equally wellwhen the 4- and/or 8-position is substituted with only a hydrogen, thusproviding 4'-primaryamino-substituted-5', (4 or 8)- dialkylpsoralens and4'-primaryamino-substituted-5'-alkylpsoralens.

Synthesis of 5' Derivatives

Under identical conditions to those described above, the4,4',8-trialkylpsoralens or the 4,4',8-trialkyl-5'-methylpsoralens canbe converted to the 5'-(ω-haloalkyl)-4,4',8-trialkylpsoralens, (hereincalled 5'-HATP), as detailed in Schematic 5, below. (See Kaufman, U.S.Pat. No. 4,294,822 and 4,298,614 for modified version).

The discussion of the conversion of 4,4',8-trialkylpsoralens to5'-primaryamino-substituted-4,4',8-trialkylpsoralens applies equallywell when the 4-, 4'- and/or 8-positions are just substituted with ahydrogen, thus providing 5'-primaryamino-substituted- dialkylpsoralensand 5'-primaryamino-substituted-alkylpsoralens, with the alkyl group(s)at the 4-, 4'- and/or 8- positions.

The discussion above of the syntheses of 4'-primaryamino- and5'-primaryamino-psoralens can be extended to the non-linear coumarins,specifically the isopsoralens or angelicins. Thus, the4'-halomethylangelicins (XIX) and the 5'-halomethylangelicins (XX) canbe prepared in a similar manner to their linear counterparts. By analogywith the synthetic pathways ##STR2## presented above one can envisionthe synthesis of 4'-(ω-amino)alkylangelicins and5'-(ω-amino)alkylangelicins where the alkyl linkage can contain one ormore oxygen or nitrogen atoms.

III. BINDING OF COMPOUNDS TO NUCLEIC ACID

The present invention contemplates binding new and known compounds tonucleic acid, including (but not limited to) viral nucleic acid andbacterial nucleic acid. One approach of the present invention to bindingphotoactivation compounds to nucleic acid is photobinding. Photobindingis defined as the binding of photobinding compounds in the presence ofphotoactivating wavelengths of light. Photobinding compounds arecompounds that bind to nucleic acid in the presence of photoactivatingwavelengths of light. The present invention contemplates methods ofphotobinding with photobinding compounds of the present invention.

One embodiment of the method of the present invention for photobindinginvolves the steps: a) providing a photobinding compound of the presentinvention; and b) mixing the photobinding compound with nucleic acid inthe presence of photoactivation wavelengths of electromagneticradiation.

The invention further contemplates a method for modifying nucleic acid,comprising the steps: a) providing photobinding compound of the presentinvention and nucleic acid; and b) photobinding the photobindingcompound to the nucleic acid, so that a compound:nucleic acid complex isformed. Without intending to be limited to any method by which thecompounds of the present invention prevent replication, it is believedthat the structure of said compound:nucleic acid complex serves toprevent replication of the nucleic acid by preventing the necessarypolymerase from acting in the region where the compound has bound.

IV. INACTIVATION OF PATHOGENS

The present invention contemplates treating a blood product with aphotoactivation compound and irradiating to inactivate contaminatingpathogen nucleic acid sequences before using the blood product.

A. Inactivation In General

The term "inactivation" is here defined as the altering of the nucleicacid of a unit of pathogen so as to render the unit of pathogenincapable of replication. This is distinct from "total inactivation",where all pathogen units present in a given sample are renderedincapable of replication, or "substantial inactivation," where most ofthe pathogen units present are rendered incapable of replication."Inactivation efficiency" of a compound is defined as the level ofinactivation the compound can achieve at a given concentration ofcompound or dose of irradiation. For example, if 100 μM of ahypothetical compound X inactivated 5 logs of HIV virus whereas underthe same experimental conditions, the same concentration of compound Yinactivated only 1 log of virus, then compound X would have a better"inactivation efficiency" than compound Y.

To appreciate that an "inactivation" method may or may not achieve"total inactivation," it is useful to consider a specific example. Abacterial culture is said to be inactivated if an aliquot of theculture, when transferred to a fresh culture plate and permitted togrow, is undetectable after a certain time period. A minimal number ofviable bacteria must be applied to the plate for a signal to bedetectable. With the optimum detection method, this minimal number is 1bacterial cell. With a sub optimal detection method, the minimal numberof bacterial cells applied so that a signal is observed may be muchgreater than 1. The detection method determines a "threshold" belowwhich the "inactivation method" appears to be completely effective (andabove which "inactivation" is, in fact, only partially effective).

B. Inactivation of Potential Pathogens

The same considerations of detection method and threshold exist whendetermining the sensitivity limit of an inactivation method for nucleicacid. Again, "inactivation" means that a unit of pathogen is renderedincapable of replication.

In the case of inactivation methods for material to be used by humans,whether in vivo or in vitro, the detection method can theoretically betaken to be the measurement of the level of infection with a disease asa result of exposure to the material. The threshold below which theinactivation method is complete is then taken to be the level ofinactivation which is sufficient to prevent disease from occuring due tocontact with the material. It is recognized that in this practicalscenario, it is not essential that the methods of the present inventionresult in "total inactivation". That is to say, "substantialinactivation" will be adequate as long as the viable portion isinsufficient to cause disease. Thus "substantially all" of a pathogen isinactivated when any viable portion of the pathogen which remaining isinsufficient to cause disease. The inactivation method of the presentinvention renders nucleic acid in pathogens substantially inactivated.In one embodiment, the inactivation method renders pathogen nucleic acidin blood preparations substantially inactivated.

Without intending to be limited to any method by which the compounds ofthe present invention inactivate pathogens, it is believed thatinactivation results from light induced binding of psoralens to pathogennucleic acid. Further, while it is not intended that the inactivationmethod of the present invention be limited by the nature of the nucleicacid; it is contemplated that the inactivation method render all formsof nucleic acid (whether DNA, mRNA, etc.) substantially inactivated.

When photoactivation compounds are used to modify nucleic acid, theinteraction of the pathogen nucleic acid (whether DNA, mRNA, etc.) withthe photoactivation compound preferably prevents replication of thepathogen, such that, if a human is exposed to the treated pathogen,infection will not result.

"Synthetic media" is herein defined as an aqueous synthetic blood orblood product storage media. In one embodiment, the present inventioncontemplates inactivating blood products in synthetic media comprising abuffered saline solution. This method reduces harm to blood products andpermits the use of much lower concentrations of photoactivationcompounds.

The psoralen photoinactivation method inactivates nucleic acid basedpathogens present in blood through a single procedure. Thus, it has thepotential to eliminate bacteria, protozoa, and viruses as well. Had aneffective decontamination method been available prior to the advent ofthe AIDS pandemic, no transfusion associated HIV transmission would haveoccurred. Psoralen-based decontamination has the potential to eliminateall infectious agents from the blood supply, regardless of the pathogeninvolved. Additionally, psoralen-based decontamination has the abilityto sterilize blood products after collection and processing, which inthe case of platelet concentrates could solve the problem of low levelbacterial contamination and result in extended storage life. Morrow J.F., et al., JAMA 266: 555-558 (1991); Bertolini F., et al., Transfusion32: 152-156 (1992).

                  TABLE 3                                                         ______________________________________                                        Viruses Photochemically Inactivated by Psoralens                              Family          Virus                                                         ______________________________________                                        Adeno           Adenovirus 2                                                                  Canine hepatitis                                              Arena           Pichinde                                                                      Lassa                                                         Bunya           Turlock                                                                       California encephalitis                                       Herpes          Herpes simplex 1                                                              herpes simplex 2                                                              Cytomegalovirus                                                               Pseudorabies                                                  Orothomyxo      Influenza                                                     Papova          SV-40                                                         Paramyxo        Measles                                                                       Mumps                                                                         Parainfluenza 2 and 3                                         Picorna.sup.1   Poliovirus 1 and 2                                                            Coxsackie A-9                                                                 Echo 11                                                       Pox             Vaccinia                                                                      Fowl Pox                                                      Reo             Reovirus 3                                                                    Blue tongue                                                                   Colorado tick fever                                           Retro           HIV                                                                           Avian sarcoma                                                                 Murine sarcoma                                                                Murine leukemia                                               Rhabdo          Vesticular stomatitis virus                                   Toga            Western equine encephalitis                                                   Dengue 2                                                                      Dengue 4                                                                      St. Louis encephalitis                                        Hepadna         hepatitis B                                                   Bacteriophage   Lambda                                                                        T2                                                            (Rickettsia)    R. akari (rickettsialpox)                                     ______________________________________                                    

A list of viruses which have been photochemically inactivated by one ormore psoralen derivatives appears in Table 3. (From Table 1 of Hanson,C. V., Blood Cells 18:7 (1992)). In the article, it was pointed out thatPiconaviruses were photoinactivated only if psoralens were presentduring virus growth. This list is not exhaustive, and is merelyrepresentative of the great variety of pathogens psoralens caninactivate. The present invention contemplates the inactivation of theseand other viruses by the compounds described herein. The compounds ofthe present invention are particularly well suited for inactivatingenvelope viruses, such as the HIV virus.

C. Selecting Photoinactivation Compounds for Inactivation of Pathogens

In order to evaluate a compound to decide if it would be useful in thephotochemical decontamination (PCD) methods of the present invention,two important properties should be considered: 1) the compound's abilityto inactivate pathogens and 2) its mutagenicity. The ability of acompound to inactivate pathogens may be determined by several methods.One technique is to perform a bacteriophage screen; an assay whichdetermines nucleic acid binding of test compounds. A screen of thistype, an r-17 screen, is described in detail in EXAMPLE 12, below. Ifthe r-17 screen shows inactivation activity, it is useful to directlytest the compound's ability to inactivate a virus. One method ofperforming a direct viral inactivation screen is described in detail inEXAMPLE 13 for cell free HIV.

The R17 bacteriophage screen is believed to be predictive of HIVinactivation efficiency, as well as the efficiency of compounds againstmany other viruses. R17 was chosen because it was expected to be a verydifficult pathogen to inactivate. It is a small, single stranded RNAphage. Without intending to be limited to any means by which the presentinvention operates, it is expected that shorter pieces of nucleic acidare harder to inactivate because they require a higher frequency offormation of psoralen adducts than do longer pieces of nucleic acid.Further, single stranded RNA pathogens are more difficult to inactivatebecause psoralens can neither intercalate between base pairs, as withdouble-stranded nucleic acids, nor form diadducts which function asinterstrand crosslinks. Thus it is expected that when inactivation ofR17 is achieved, these same conditions will cause the inactivation ofmany viruses and bacteria.

The cell free HIV screen complements the r-17 screen by affirming that agiven compound which has tested positive in r-17 will actually workeffectively to inactivate viruses. Thus, if a compound shows activity inthe r-17 screen, it is next tested in the viral inactivation screen.

The second property that is important in testing a compound for use inmethods of the present invention is mutagenicity. The most widely usedmutagen/carcinogen screening assay is the Ames test. This assay isdescribed by D. M. Maron and B. N. Ames in Mutation Research 113: 173(1983) and a specific screen is described in detail in Example 17,below. The Ames test utilizes several unique strains of Salmonellatyphimurium that are histidine-dependent for growth and that lack theusual DNA repair enzymes. The frequency of normal mutations that renderthe bacteria independent of histidine (i.e., the frequency ofspontaneous revertants) is low. The test allows one to evaluate theimpact of a compound on this revertant frequency.

Because some substances are not mutagenic by themselves, but areconverted to a mutagen by metabolic action, the compound to be tested ismixed with the bacteria on agar plates along with the liver extract. Theliver extract serves to mimic metabolic action in an animal. Controlplates have only the bacteria and the extract.

The mixtures are allowed to incubate. Growth of bacteria (if any) ischecked by counting colonies. A positive Ames test is one where thenumber of colonies on the plates with mixtures containing the compoundsignificantly exceeds the number on the corresponding control plates.

When known carcinogens are screened in this manner with the Ames test,approximately ninety percent are positive. When known noncarcinogens aresimilarly tested, approximately ninety percent are negative.

A new compound (X) can be evaluated as a potential bloodphotodecontamination compound, as shown in Table 4, below. X isinitially evaluated in Step I. X is screened in the r-17 assay atseveral different concentrations between 4 and 320 μM, as explained inEXAMPLE 12. If the compound shows inactivation activity greater than 1log inactivation of r-17 (log kill) in the r-17 screen at anyconcentration, the compound is then screened in the cell free HIV assay,as explained in EXAMPLE 13. If the compound shows inactivation activitygreater than 1 log inactivation of HIV (log kill) in the cell free HIVassay, the compound and AMT are then screened in the Ames assay.Finally, if the compound shows lower mutagenicity in the Ames assay thandoes AMT, the new compound is identified as a useful agent forinactivation of pathogens.

                  TABLE 4                                                         ______________________________________                                                                       IN-                                            STEP  SCREEN     RESULT        TERPRETATION                                   ______________________________________                                        1     r-17       >1 log kill by any                                                                          potential PCD com-                                              concentration pound, go to step 2                                             <1 log kill   compound is ineffec-                                                          tive as an inactiva-                                                          tion treatment                                 2     Viral      >1 log kill by any                                                                          potential PCD com-                                   Inactivation                                                                             concentration pound, go to step 3                                             <1 log kill   compound is ineffec-                                                          tive as an inactiva-                                                          tion treatment                                 3     Ames       less mutagenic                                                                              useful agent for                                                than AMT      PCD                                            ______________________________________                                    

By following these instructions, a person can quickly determine whichcompounds would be appropriate for use in methods of the presentinvention.

D. Delivery of Compounds for Photoinactivation

The present invention contemplates several different formulations androutes by which the compounds described herein can be delivered in aninactivation method. This section is merely illustrative, and notintended to limit the invention to any form or method of introducing thecompound.

The compounds of the present invention may be introduced in aninactivation method in several forms. The compounds may be introduced asan aqueous solution in water, saline, a synthetic media such as"Sterilyte™ 3.0" (contents set forth at the beginning of theExperimental section, below) or a variety of other media. The compoundscan further be provided as dry formulations, with or without adjuvants.

The new compounds may also be provided by many different routes. Forexample, the compound may be introduced to the reaction vessel, such asa blood bag, at the point of manufacture. Alternatively, the compoundmay be added to the material to be sterilized after the material hasbeen placed in the reaction vessel. Further, the compounds may beintroduced alone, or in a "cocktail" or mixture of several differentcompounds.

V. PRESERVATION OF BIOCHEMICAL PROPERTIES OF MATERIAL TREATED

When treating blood products to be used in vivo, two factors are ofparamount importance in developing methods and compounds to be used.First, one must ask whether the process or the compounds used alter thein vivo activity of the treated material. For example, platelettransfusion is a well established efficacious treatment for patientswith thrombocytopenic bleeding. However, if the decontaminationtreatment used greatly reduces the platelets clotting activity, then thetreatment has no practical value. Psoralens are useful in inactivationprocedures, because the reaction can be carried out at temperaturescompatible with retaining biochemical properties of blood and bloodproducts. Hanson, C. V., Blood Cells 18:7 (1992). But not all psoralensor methods will decontaminate without significantly lowering thebiological activity of the decontaminated material. Previous compoundsand protocols have necessitated the removal of molecular oxygen from thereaction before and during exposure to light, to prevent damage to bloodproducts from oxygen radicals produced during irradiation. See L. Lin etal., Blood 74:517 (1989); U.S. Pat. No. 4,727,027, to Wiesehahn. Thepresent invention may be used to decontaminate blood products, in thepresence of oxygen, without destroying the activity for which theproducts are prepared. Further, with methods of the present invention,there is no need to reduce the concentration of molecular oxygen. Thepresent invention contemplates that in vivo activity of a blood productis not destroyed or significantly lowered if a sample of blood productwhich is decontaminated by methods of the present invention tests aswould a normally functioning sample of blood product in known assays forblood product function. For example, where platelets are concerned, invivo activity is not destroyed or significantly lowered if pH of theplatelets are substantially the same in platelets treated by the methodsof the present invention and stored 5 days as they are in untreatedsamples stored for 5 days. "Substantially the same" pH means that thevalues fall within the range of error surrounding that particular datapoint.

The present invention contemplates a preferred way of protecting bloodand blood products during irradiation by introducing a synthetic mediacomprising a buffered saline solution for the period of irradiation andany storage thereafter. In a preferred embodiment of the presentinvention, dibasic phosphate is used as a buffer. Dibasic phosphatecontains both singly charged ions and a doubly charged ions.

The second factor is whether the compounds used are toxic or mutagenicto the patient treated. A "compound displaying low mutagenicity" isdefined as a compound which is less mutagenic than AMT when it is testedat concentrations below 250 μM in the Ames assay, described in theExperimental section, below. The inactivation compounds and methods ofthe present invention are especially useful because they display theunlinking of pathogen inactivation efficiency from mutagenicity. Thecompounds exhibit powerful pathogenic inactivation without a concomitantrise in mutagenicity. The commonly known compounds tested inphotoinactivation protocols, such as AMT, appear to exhibit a linkbetween pathogen inactivation efficiency and mutagenetic action thatuntil now seemed indivisible.

While it is not intended that the present invention be limited to anytheory by which pathogen inactivation efficiency is unlinked frommutagenicity, it is postulated that unlinking occurs as a result of thelength of the groups substituted on the psoralen, and the location ofcharges on the compounds. It is postulated that positive charges on oneor both ends of mutagenic compounds have non-covalent interactions withthe phosphate backbone of DNA. These interactions are presumed to occurindependent of the presence of light (called "dark binding"). In theory,the psoralen thereby sterically blocks polymerase from opening up theDNA, causing mutagenicity. In contrast, compounds of the presentinvention carry a positive or neutral charge on a long substitute group.These substituted groups form a steric barrier during dark binding thatis much easier to free from the DNA, permitting polymerase to pass. Thusno mutagenicity results.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); Kg(kilograms); L (liters); mL (milliliters); μL(microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); J(Joules, also watt second, note that in FIGS. 11, 13-22 Joules or Jrefers to Joules/cm²); °C. (degrees Centigrade); TLC (Thin LayerChromatography); EAA (ethyloacetoacetate); EtOH (ethanol); HOAc (aceticacid); W (watts); mW (milliwatts); NMR (Nuclear Magnetic Resonance;spectra obtained at room temperature on a Varian Gemini 200 MHz FourierTransform Spectrometer); m.p. (melting point); UV (ultraviolet light);THF (tetrahydrofuran); DMEM (Dulbecco's Modified Eagles Medium); FBS(fetal bovine serum); LB (Luria-Bertani); EDTA (ethelene diaminetetracidic acid); Phorbol Myristate Acetate (PMA); phosphate bufferedsaline (PBS); BSA (bovine serum albumin); PCR (polymerase chainreaction). Further, in the examples describing synthesis of compounds ofthe present invention, yields presented are for the preceeding steponly, rather than for the entire synthesis.

For ease of reference, some compounds of the present invention have beenassigned a number from 1-18. The reference numbers are assigned in TABLE2, Their structures appear in FIGS. 10A-10F. The reference numbers areused throughout the experimental section.

When isolating compounds of the present invention in the form of an acidaddition salt, the acid is preferably selected so as to contain an anionwhich is non-toxic and pharmacologically acceptable, at least in usualtherapeutic doses. Representative salts which are included in thispreferred group are the hydrochlorides, hydrobromides, sulphates,acetates, phosphates, nitrates, methanesulphonates, ethanesulphonates,lactates, citrates, tartrates or bitartrates, and maleates. Other acidsare likewise suitable and may be employed as desired. For example,fumaric, benzoic, ascorbic, succinic, salicylic, bismethylenesalicylic,propionic, gluconic, malic, malonic, mandelic, cinnamic, citraconic,stearic, palmitic, itaconic, glycolic, benzenesulphonic, and sulphamicacids may also be employed as acid addition salt-forming acids.

One of the examples below refers to HEPES buffer. This buffer contains8.0 g of 137 mM NaCl, 0.2 g of 2.7 mM KCl, 0.203 g of 1 mM MgCl₂ (6H₂O), 1.0 g of 5.6 mM glucose, 1.0 g of 1 mg/ml Bovine Serum Albumin (BSA)(available from Sigma, St. Louis, Mo.), and 4.8 g of 20 mM HEPES(available from Sigma, St. Louis, Mo.).

In one of the examples below, phosphate buffered synthetic media isformulated for platelet treatment. This can be formulated in one step,resulting in a pH balanced solution (e.g. pH 7.2), by combining thefollowing reagents in 2 liters of distilled water:

    ______________________________________                                        Preparation of Sterilyte ™ 3.0                                                      Formula W.                                                                             mMolarity  Grams/2 Liters                                   ______________________________________                                        NaAcetate*3H.sub.2 O                                                                     136.08     20         5.443                                        Glucose    180.16     2          0.721                                        D-mannitol 182.17     20         7.287                                        KCl        74.56      4          0.596                                        NaCl       58.44      100        11.688                                       Na.sub.3 Citrate                                                                         294.10     10         5.882                                        Na.sub.2 HPO.sub.4 *7H.sub.2 O                                                           268.07     14.46      7.752                                        NaH.sub.2 PO.sub.4 *H.sub.2 O                                                            137.99     5.54       1.529                                        MgCl.sub.2 *6H.sub.2 O                                                                   203.3      2          0.813                                        ______________________________________                                    

The solution is then mixed, sterile filtered (0.2 micron filter) andrefrigerated.

Another synthetic media contains the following reagents:

    ______________________________________                                        Preparation of synthetic media + phosphate                                             Formula W.                                                                             mMolarity  Grams/Liters                                     ______________________________________                                        NaAcetate*3H.sub.2 O                                                                     136.08     30         4.08                                         NaCl       58.44      86         5.02                                         Citrate*2H.sub.2 O                                                                       294.10     10         2.94                                         Na.sub.2 HPO.sub.4                                                                       142.07     19.8       2.81                                         NaH.sub.2 PO.sub.4 *H.sub.2 O                                                            137.99     6.2        0.858                                        ______________________________________                                    

The Polymerase Chain Reaction (PCR) is used in one of the examples tomeasure whether viral inactivation by some compounds was complete. PCRis a method for increasing the concentration of a segment of a targetsequence in a mixture of genomic DNA without cloning or purification.See K. B. Mullis et al., U.S. Pat. Nos. 4,683,195 and 4,683,202, herebyincorporated by reference. This process for amplifying the targetsequence consists of introducing a large excess of two oligonucleotideprimers to the DNA mixture containing the desired target sequence,followed by a precise sequence of thermal cycling in the presence of aDNA polymerase. The two primers are complementary to their respectivestrands of the double stranded target sequence. To effect amplification,the mixture is denatured and the primers then to annealed to theircomplementary sequences within the target molecule. Following annealing,the primers are extended with a polymerase so as to form a new pair ofcomplementary strands. The steps of denaturation, primer annealing, andpolymerase extension can be repeated many times (i.e. denaturation,annealing and extension constitute one "cycle;" there can be numerous"cycles") to obtain a high concentration of an amplified segment of thedesired target sequence. The length of the amplified segment of thedesired target sequence is determined by the relative positions of theprimers with respect to each other, and therefore, this length is acontrollable parameter. By virtue of the repeating aspect of theprocess, the method is referred to by the inventors as the "PolymeraseChain Reaction". Because the desired amplified segments of the targetsequence become the predominant sequences (in terms of concentration) inthe mixture, they are said to be "PCR amplified".

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g. hybridization with a labelled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³² p labelled deoxynucleotide triphosphates, e.g. dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide sequence can be amplified with the appropriate set ofprimer molecules.

The PCR amplification process is known to reach a plateau concentrationof specific target sequences of approximately 10⁻⁸ M. A typical reactionvolume is 100 μl, which corresponds to a yield of 6×10¹¹ double strandedproduct molecules.

PCR is a polynucleotide amplification protocol. The amplification factorthat is observed is related to the number (n) of cycles of PCR that haveoccurred and the efficiency of replication at each cycle (E), which inturn is a function of the priming and extension efficiencies during eachcycle. Amplification has been observed to follow the form E^(n), untilhigh concentrations of PCR product are made. At these highconcentrations (approximately 10⁻⁸ M/l) the efficiency of replicationfalls off drastically. This is probably due to the displacement of theshort oligonucleotide primers by the longer complementary strands of PCRproduct. At concentrations in excess of 10⁻⁸ M, the rate of the twocomplementary PCR amplified product strands finding each other duringthe priming reactions become sufficiently fast that this occurs beforeor concomitant with the extension step of the PCR procedure. Thisultimately leads to a reduced priming efficiency, and therefore, areduced cycle efficiency. Continued cycles of PCR lead to decliningincreases of PCR product molecules. PCR product eventually reaches aplateau concentration.

The sequences of the polynucleotide primers used in this experimentalsection are as follows:

DCD03: 5' ACT AGA AAA CCT CGT GGA CT 3'

DCD05: 5' GGG AGA GGG GAG CCC GCA CG 3'

DCD06: 5' CAA TTT CGG GAA GGG CAC TC 3'

DCD07: 5' GCT AGT ATT CCC CCG AAG GT 3'

With DCD03 as a common forward primer, the pairs generate amplicons oflength 127, 327, and 1072 bp. These oligos were selected from regionsthat are absolutely conserved between 5 different dHBV isolates (DHBV1,DHBV3, DHBV16, DHBV22, and DHBV26) as well as from heron HBV (HHBV4).

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

EXAMPLE 1

As noted above, the present invention contemplates devices and methodsfor the photoactivation of photoreactive nucleic acid binding compounds.In this example, a photoactivation device is described fordecontaminating blood products according to the method of the presentinvention. This device comprises: a) means for providing appropriatewavelengths of electromagnetic radiation to cause photoactivation of atleast one photoreactive compound; b) means for supporting a plurality ofblood products in a fixed relationship with the radiation providingmeans during photoactivation; and c) means for maintaining thetemperature of the blood products within a desired temperature rangeduring photoactivation.

FIG. 6 is a perspective view of one embodiment of the device integratingthe above-named features. The figure shows an opaque housing (100) witha portion of it removed, containing an array of bulbs (101) above andbelow a plurality of representative blood product containing means (102)placed between plate assemblies (103, 104) which filter certainwavelengths of light. The plate assemblies (103, 104) are described morefully, subsequently.

The bulbs (101), which are connectable to a power source (not shown),serve as a source of electromagnetic radiation. While not limited to theparticular bulb type, the embodiment is configured to accept an industrystandard, dual bipin lamp.

The housing (100) can be opened via a latch (105) so that the bloodproduct can be placed appropriately. As shown in FIG. 6, the housing(100), when closed, completely contains the irradiation from the bulbs(101). During irradiation, the user can confirm that the device isoperating by looking through a safety viewport (106) which does notallow transmission of ultraviolet light to the user.

The housing (100) also serves as a mount for several electroniccomponents on a control board (107), including, by way of example, amain power switch, a count down timer, and an hour meter. Forconvenience, the power switch can be wired to the count down timer whichin turn is wired in parallel to an hour meter and to the source of theelectromagnetic radiation. The count down timer permits a user to presetthe irradiation time to a desired level of exposure. The hour metermaintains a record of the total number of radiation hours that areprovided by the source of electromagnetic radiation. This featurepermits the bulbs (101) to be monitored and changed before their outputdiminishes below a minimum level necessary for rapid photoactivation.

FIG. 7 is a cross-sectional view of the device shown in FIG. 6 along thelines of 2--2. FIG. 7 shows the arrangement of the bulbs (101) with thehousing (100) opened. A reflector (108A, 108B) completely surrounds eacharray of bulbs (101). Blood product containing means (102) are placedbetween upper (103) and lower (104) plate assemblies (e.g. BK-7 glass,Shott Glass Technologies, Inc., Duryea, Pa.). Each plate assembly iscomprised of an upper (103A, 104A) and lower (103B, 104B) plates. Theplate assemblies (103, 104) are connected via a hinge (109) which isdesigned to accommodate the space created by the blood productcontaining means (102). The upper plate assembly (103) is brought torest just above the top of the blood product containing means (102)supported by the lower plate (104B) of the lower plate assembly (104).

Detectors (110A, 110B, 110C, 110D) may be conveniently placed betweenthe plates (103A, 103B, 104A, 104B) of the plate assemblies (103, 104).They can be wired to a printed circuit board (111) which in turn iswired to the control board (107).

FIG. 8 is a cross-sectional view of the device shown in FIG. 6 along thelines of 3--3. Six blood product containing means (102) (e.g. Teflon™platelet unit bags) are placed in a fixed relationship above an array ofbulbs (101). The temperature of the blood product can be controlled viaa fan (112) alone or, more preferably, by employing a heat exchanger(113) having cooling inlet (114) and outlet (115) ports connected to acooling source (not shown).

FIG. 9 is a cross-sectional view of the device shown in FIG. 6 along thelines of 4--4. FIG. 9 more clearly shows the temperature controlapproach of a preferred embodiment of the device. Upper plate assemblyplates (103A, 103B) and lower plate assembly plates (104A, 104B) eachcreate a temperature control chamber (103C, 104C), respectively. The fan(112) can circulate air within and between the chambers (103C, 104C).When the heat exchanger (113) is employed, the circulating air is cooledand passed between the plates (103A, 103B, 104A, 104B).

EXAMPLE 2 Synthesis of 4'-Bromomethyl-4,5',8-trimethylpsoralen

In this example, the three step synthesis of4'-Bromomethyl-4,5',8-trimethylpsoralen is described. This synthesis isperformed without a bromomethylation step, making it safer than knownmethods of synthesis.

Step 1:

3-Chloro-2-butanone (29.2 mL, 0.289 mol) was added to a mechanicallystirred suspension of 7-hydroxy-4,8-dimethylcoumarin (50.00 g, 0.263mol) and powdered K₂ CO₃ (54 g, 0.391 mol) in acetone (500 mL). Theslurry was refluxed for 15 hours, after which the solvent was strippedoff. To remove the salt, the solid was stirred in 1.2 L of water,filtered, and rinsed with water until the pH of the mother liquor wasneutral (pH 5-7). The brown filtrate was dissolved in boiling methanol(150 mL), allowed to cool to room temperature to form a thick paste andrinsed with ice cold methanol to remove most of the brown impurity,giving 4,8-dimethyl-7-(1-methyl-2-oxo)propyloxy-coumarin (67.7 g, 99.0%yield) as an off-white solid, melting point 95°-96° C. NMR: d 1.57 (d,J=6.7 Hz, 3H), 2.19 (s, 3H), 2.39 (s, 6H), 4.73(q, J=6.9 Hz, 1H), 6.17(s, 1H), 6.63 (d, J=8.8 Hz, 1H), 7.38 (d, J=8.9 Hz, 1H).

Step 2:

A suspension of 4,8-dimethyl-7-(1-methyl-2-oxo)propyloxy-coumarin (67.5g, 0.260 mol), 10% aqueous NaOH (114 mL, 0.286 mol) and water (900 mL)was heated for 2-4 hours at 70°-85° C. The mixture was then allowed tocool to room temperature. The solid was filtered, and then rinsed withchilled water (1.5 L) until the mother liquor became colorless and pHwas neutral (pH 5-7). The product was air and vacuum dried to give4,4',5',8-tetramethylpsoralen (56.3 g, 89.5%) as a white solid, mp197°-199° C. NMR: d 2.19 (s, 3H), 2.42 (s, 3H), 2.51 (s, 3H), 2.56 (s,3H), 6.23 (s, 1H), 7.40 (s, 1H).

Step 3:

Dry 4,4',5',8-tetramethylpsoralen (10.00 g, 41.3 mmol) was dissolved inmethylene chloride (180 mL) at room temperature. N-Bromosuccinimide(8.09 g, 45.3 mmol) was added and the reaction mixture and stirred 4.5hours. The solvent was completely removed and the resulting solid wasstirred with water (200 mL) for 0.5-1 h, filtered and cold trituratedwith additional water (approximately 500 mL) to remove the succinimideby-product. The crude product (i.e.4'-bromomethyl-4,5',8-trimethylpsoralen) was dried in a vacuumdessicator with P₂ O₅ then recrystallized in a minimum amount of boilingtoluene (200-300 mL) to give 4'-bromomethyl-4,5',8-trimethylpsoralen(10.2 g), a pale yellow solid. The mother liquor was stripped andrecrystallized again with toluene (60 mL) to give a second crop ofproduct (1.08 g, combined yield=85.1%, >99% purity by NMR), mp 206°-207°C. NMR: d 2.50 (s, 3H), 2.54 (d, J=1.2 Hz, 3H), 2.58 (s, 3H), 4.63 (s,2H), 6.28 (apparent q, J=1.3 Hz, 1H), 7.59 (s, 1H).

EXAMPLE 3 Synthesis of 5'-bromomethyl-4,4',8-trimethylpsoralen

In this example, a three step synthesis of5'-bromomethyl-4,4',8-trimethylpsoralen is described. Like the synthesisdescribed in Example 2, this method is improved upon previously knownsynthesis schemes because it does not require bromomethylation.

4,4',5',8-Tetramethylpsoralen (2.33 g, 9.59 mmol), (synthesis describedin Example 2, Steps 1 and 2), was refluxed in carbon tetrachloride (100mL) until it dissolved. N-Bromosuccinimide (1.88 g, 10.5 mmol) andbenzoyl peroxide (80 mg) were then added and the mixture was refluxedfor 15 hours. Upon cooling to room temperature methylene chloride (100mL) was added to dissolve the solid and the solution was washed withwater (4×150 mL), then brine, and dried with anhydrous Na₂ SO₄. Thesolvent was stripped off to give a mixture of5'-bromomethyl-4,4',8-trimethylpsoralen,4'-bromomethyl-4,5',8-trimethylpsoralen, and4',5'-bis(bromomethyl)-4,8-dimethylpsoralen in a ratio of 55/25/20respectively as determined by ¹ H NMR (3.0 g, crude product). ¹ H NMR of5'-bromomethyl compound: d 2.29 (s, 3H), 2.52 (d, J=1.2 Hz, 3H), 2.60(s, 3H), 4.64 (s, 2H), 6.27 (apparent d, J=1.2 Hz, 1H), 7.51 (s,1H). ¹ HNMR of 4',5'-bis(bromomethyl) compound: d 2.54 (d, J=1.1 Hz, 3H), 2.60(s, 3H), 4.65 (s, 4H), 6.30 (apparent q, J=1.1 Hz, 1H), 7.67 (s, 1H).

EXAMPLE 4 Synthesis of 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralenHydrochloride (Compound 2) and Related Compounds (Compound 4)

In this example, two methods of synthesis of Compound 2 are described.The first method was performed as follows:

Step 1:

4'-Bromomethyl-4,5',8-trimethylpsoralen (3.09 g, 9.61 mmol), (synthesisdescribed in Example 2), and N-(2-hydroxyethyl)phthalimide (4.05 g, 21.2mmol) were stirred in dry dimethylformamide (65 mL). Dry N₂ gas wasbubbled gently into the reaction mixture. The reaction mixture washeated to 100° C. for 4.5 hours then allowed to cool to room temperatureand put in the freezer for several hours. The crystalline product wasfiltered and washed with MeOH followed by H₂ O. The solid was furthertritutrated with MeOH (100 mL) to remove the impurities. The crudeproduct was air dried and dissolved in CHCl₃ (150 mL). Activated carbonand silica gel were added to decolorize and the CHCl₃ was completelyremoved. The resulting white product,4'-[4-(N-phthalimido)-2-oxa]butyl-4,5',8-trimethylpsoralen (1.56 g,yield 37.5%) was ≧99% pure both by NMR and HPLC; mp 224°-225° C. NMR(CDCl₃) δ2.37 (s,3H); 2.47 (s, 3H); 2.48 (s, 3H); 3.78 (s,4H); 4.59(s,2H); 6.22 (s, 1H);7.42 (s,1H); 7.50 (m, 4H).

Step 2:

4'-[4-(N-phthalimido)-2-oxa]butyl-4,5',8-trimethylpsoralen (1.56 g, 3.61mmol) was suspended in tetrahydrofuran (75 mL) at room temperature.Methylamine (40% aqueous solution, 25 mL, 290 mmol) was added to thesuspension and stirred overnight. The solvent and methylamine werecompletely removed. The resulting solid was taken up in 0.3N HCl aqueoussolution (25 mL). The acid suspension was rinsed three times with 40 mLCHCl3 then taken to pH 11 with 20% aqueous NaOH. CHCl₃ (3×60 mL) wasused to extract the product (i.e.4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen) from the basifiedlayer. The combined CHCl₃ layers were washed with H₂ O (100 mL) followedby brine (100 mL) then dried over anhydrous Na₂ SO₄ and concentrated togive 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen, mp 139°-141° C.Purity was greater than 99% by NMR. NMR (CDCl₃) δ2.50 (s, 6H); 2.58(s,3H); 2.90 (t, J=5.27 Hz, 2H); 3.53 (t, J=5.17 Hz, 2H); 4.66 (s, 2H);6.25 (s, 1H); 7.61 (s, 1H). The4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen was dissolved inabsolute ethanol (150 mL), a 1.0M solution of HCl in ether (10 mL) wasadded and the suspension was cooled in the freezer overnight. Afterfiltration and washing with ether, the solid was vacuum dried to givepale yellow crystals (0.76 g, yield 62%), mp 235°-236° C.

Alternatively, Step 2 may be performed using either hydrazine orbutylamine rather than methylamine. The method which uses butylamine ispreferred for larger scale syntheses because, while an excess ofmethylamine is needed due to volatization, the same is not true forbutylamine. The method using butylamine was carried out as follows: 28.3g phthalimide has been deprotected with n-butylamine in propanol. Thecrude reaction solution is then treated with HCl to precipitate out theproduct. Thus the reaction mixture in 285 mL of 1-propanol was treatedwith HCl gas to pH 2. The mixture was stirred at 5° C. for 0.5 hours,then filtered and washed with cold solvent (3×15 mL) to afford 20.5 g ofcrude 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen (92% yield).

The method using hydrazine was carried out as follows: The phthalimideprecursor (6 mol) was deprotected with hydrazine and after concentrationand acid-base extractions the crude amine was obtained in 30 L ofethylene dichloride. To this was added HCl gas (0.14 Kg) via dispersiontuve over 40 minutes maintaining the temperature at 15°-25° C. Theresultant slurry was stirred an additional 1 hour. The solids werecollected on a Buchner funnel. Upon drying in an air dryer at 80° C. for2 hours, 0.945 kg of crude4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen was obtained. 0.94 kgof product in a mixture of 7.5 kg of isopropanol and 1.88 kg of waterwas refluxed for 30 minutes then hot filtered. The solution was cooledto room temperature over 1 hour, then chilled to 15°-20° C. for 0.5hours. The solids were collected on a Buchner funnel, then washed withcold isopropanol (0.3 L). The wet solids were transferred to glass traysand dried under vacuum (>28 minutes) at approximately 75° C. for 11.5hours. Moisture content was 0.5%. Yield was 0.758 kg (81% yield). The4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen was analytically pure.Residual isopropanol about 1700 ppm by NMR.

The first method is a preferred embodiment of the present inventionbecause of its high yield and purity.

The second method starts with the preparation of4'-chloromethyl-4,5',8-trimethylpsoralen from commercially available4,5',8-trimethylpsoralen, as described above. The synthesis of4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen hydrochloride isachieved in four (4) steps:

STEP 1:

4'-Chloromethyl-4,5',8-trimethylpsoralen (550 mg, 1.99 mmol) andethylene glycol (6.8 ml, 121.9 mmol) were heated in acetone (6 mL) to50°-60° C. for 3.5 hrs. After 2 hrs heating, the white suspension hadturned to a clear light yellow solution. The acetone and ethylene glycolwere removed on the rotoevaporator and water (50 mL) was added to theresidue. The resultant suspension was filtered, washed with cold waterthen dried in the vacuum oven to give 574 mg (96%) of4'-(4-hydroxy-2-oxa)butyl-4,5',8-trimethylpsoralen; NMR (CDCl₃) δ2.51(s, 6H); 2.58 (s, 3H); 3.62 (t, J=4.5 Hz, 2H); 3.78 (t, J=4.9 Hz, 2H);4.70 (s, 2H); 6.26 (d, J=1.1 Hz, 1H); 7.61 (s, 1H).

STEP 2:

4'-(4-Hydroxy-2-oxa)butyl-4,5',8-trimethylpsoralen (574 mg, 1.9 mmol)was dissolved in CH₂ Cl₂ (6 mL) under N₂ at <10° C. Triethylamine (359mg, 3.55 mmol) was added. Methanesulfonyl chloride (305 mg, 266 mmol)was dropped in slowly keeping the temperature below 10° C. Afteraddition was completed the mixture was stirred for 15 more minutes andthen it was stirred at room temperature for 10 hours. To the reactedsuspension CH₂ Cl₂ (45 mL) was added and the mixture was washed withwater (3×20 mL), then dried over anhydrous Na₂ SO₄. Concentration at<30° C. followed by vacuum drying gave4'-[(4-methanesulfonyloxy-2-oxa)butyl-4,5',8-trimethylpsoralen as ayellow solid (706 mg, 98%), mp 138°-140° C. NMR δ2.51 (s, 3H); 2.52 (d,3H); 2.58 (s, 3H); 2.99 (s, 3H); 3.77 (m,2H); 4.39 (m, 2H); 4.71 (s,2H); 6.26(s, 1H); 7.62 (s, 1H).

STEP 3:

4'-[(4-Methanesulfonyloxy-2-oxa)butyl-4,5',8-trimethylpsoralen (706 mg,1.86 mmol) and sodium azide (241 mg, 3.71 mmol) were refluxed in 95%ethyl alcohol (5 mL) for 8 hours. The reaction solution was cooled andcold water (55 mL) was added. The off-white solid was filtered andwashed with cold water. Upon vacuum drying, the azide (i.e.4'-(4-Azido-2-oxa)butyl-4,5',8-trimethylpsoralen) was obtained as alight yellowish solid (575 mg, 95%), mp 105°-106° C. NMR: δ2.51 (s, 6H);2.58 (s, 3H); 3.41 (t, J=4.9 Hz, 2H); 3.67 (apparent t, J=4.9 Hz, 2H);4.70 (s, 2H); 6.26 (s, 1H); 7.66 (s, 1H).

STEP 4:

The 4'-(4-Azido-2-oxa)butyl-4,5',8-trimethylpsoralen (1.65 g, 5.03 mmol)was dissolved in tetrahydrofuran (10 mL). Triphenylphosphine (1.59 g,6.08 mmol) and six drops of water were added to the foregoing solution.After stirring at room temperature overnight, the light yellow solutionwas concentrated. The residue was dissolved in CHCl₃ (90 mL) andextracted with 0.3N aqueous HCl (30 mL, then 2×5 mL). Combined HCllayers was carefully treated with K₂ CO₃ until saturated. The basesolution was extracted with CHCl₃ (3×60 mL). Combined CHCl₃ layers werewashed with 60 mL of water, 60 mL of brine and dried over anhydrous Na₂SO₄. Upon concentration and vacuum drying the amine (i.e. was obtainedas a yellow solid (1.25 g, 82%), mp 139°-141° C.; NMR δ2.48 (s, 6H);2.55 (s, 3H); 2.89 (t, J=6 Hz, 2H); 3.52 (t, J=6 Hz, 2H); 4.64 (s, 2H);6.22 (s, 1H); 7.59 (s, 1H).

The amine was dissolved in absolute ethanol (40 mL) and 20 mL of 1N HClin ethyl ether was added. After sitting at 5° C. overnight, theprecipitate was filtered and rinsed with ether to give 1.25 g ofCompound 2, mp 236° C. (decomp). ¹³ C NMR: 8.54, 12.39, 19.18, 38.75,62.26, 65.80, 108.01, 112.04, 112.42, 112.97, 116.12, 125.01, 148.76,153.97, 154.37, 155.76, 160.34.

Anal. Calculated for C₁₇ H₂₀ ClNO₄ : C, 60.45: H,5.97; N, 4.15. Found:C, 60.27; H, 5.88; N, 4.10.

Similarly prepared, by reacting 4'-CMT with 1,3-propanediol comparablyto Step 1 and proceeding analagously through Step 4, was4'-(5-amino-2-oxa)pentyl-4,5',8-trimethylpsoralen, (Compound 4), m.p.212°-214° C. (decomposed). NMR of the free base: δ1.73 (pent, J=6.4 Hz,2H), 2.45(s, 6H), 2.51 (s, 3H), 2.78 (t,J=6.8 Hz, 2H), 3.54 (t, J=6.2Hz, 2H), 4.59 (s,2n), 6.18 (s, 1H), 7.54 (s, 1H).

EXAMPLE 5 Synthesis of 5'-(4-Amino-2-oxa)butyl-4,4',8-trimethylpsoralen(Compound 18)

This example describes the synthesis of Compound 18. To a stirredsolution of N-methylformanilide (16.0 mL, 0.134 mol) in acetonitrile(130 mL) was added phosphorus oxychloride (12.5 mL, 0.134 mol), then4,4',8-trimethylpsoralen (5.0 g, 21.9 mmol) (described in McLeod, etal., Tetrahedron Letters No. 3:237 (1972)). The temperature was keptbetween 0°-10° C. during addition of the psoralen by use of an ice/waterbath. The slurry was stirred at 50° C. for 2 days protected frommoisture with a drierite drying tube. The reaction mix was allowed tocool to room temperature, then chilled in an ice/water bath. Theacetonitrile was decanted off, then ice/water (150 mL) was added to theorange slurry and stirred for 1 h. The orange solid was filtered off andrinsed with chilled water, then chilled acetonitrile. The crude productwas recrystallized and charcoal decolorized in dichloroethane (600 mL)to give 4,4',8-trimethyl-5'-psoralencarboxaldehyde (3.59 g, 64.0%) as apale yellow-orange solid, sublimes ≧250° C., decomp. >300° C. ¹ H NMR(CDCl₃): 2.54 (d, J=1 Hz, 3H), 2.64 (s, 3H), 2.68 (s, 3H), 6.32(apparent d, J=1 Hz, 1H), 7.75 (s, 1H), 0.07 (s, 1H).

4,4',8-trimethyl-5-psoralencarboxaldehyde (7.50 g, 29.3 mmol) wasstirred in 200 proof EtOH (250 mL). Sodium borohydride was added and theslurry was stirred overnight. Ice water (150 mL) and 10% aq NaCO₃ (50mL) were added to quench the reaction. After stirring for 45 min, theprecipitate was filtered off and rinsed with water until the filtratewas neutral (pH 5-7). The product was dried in a vacuum dessicator withP₂ O₅ to give 5'-hydroxymethyl-4,4',8-trimethylpsoralen (7.46 g, 98.5%)as a pale yellow solid, mp 244°-245° C. ¹ H NMR (CDCl₃): 1.97 (t, J=6Hz, 1H), 2.31 (s, 3H), 2.51 (d, J=1 Hz, 3H), 2.58 (s, 3H), 4.79 (d, J=6Hz, 2H), 6.25 (apparent d, J=1 Hz, 1H), 7.49 (s, 1H).

To a stirred, ice/water chilled slurry of5'-hydroxymethyl-4,4',8-trimethylpsoralen (15.42 g, 59.7 mmol) indichloroethane (500 mL) was added phosphorus tribromide (6.17 mL, 65.7mmol) dropwise. The reaction was protected from moisture and allowed tostir overnight at room temperature. The mixture was then stirred with300 mL ice/water for 1 h. The solid was filtered off, dried, dissolvedin hot toluene, filtered through fluted filter paper and stripped togive 5'-bromomethyl-4,4',8-trimethylpsoralen (3.43 g). The reactionsolvents (dichloroethane and water) were separated and the aqueous layerwas extracted three times with dichloroethane. The organic layers werecombined, rinsed with brine then dried (anhyd Na₂ SO₄) and strippedunder vacuum to give the bulk of the product,5'-bromomethyl-4,4',8-trimethylpsoralen, (13.13 g, combined yield of86.4%), as a pale yellow solid, mp 201°-202° C. ¹ H NMR (CDCl₃): 2.29(s, 3H), 2.52 (d, J=1 Hz, 3H), 2.60 (s, 2H), 4.64 (s, 2H), 6.27(apparentd, J=1 Hz, 1H), 7.51 (s, 1H).

N-Hydroxyethylphthalimide (3.00 g, 15.5 mmol) was dissolved in DMF (5mL) at 60°-64° C. while N₂ was bubbled into the solution. Sodium iodide(0.01 g, 0.067 mmol) and 5'-bromomethyl-4,4',8-trimethylpsoralen (1.00g, 3.11 mmol) were added and the slurry was stirred under theseconditions overnight. The thick yellow reaction mixture was allowed tocool to room temperature, chilled in an ice/water bath, filtered andrinsed with ice cold MeOH to give crude product (1 g). The solid wasrecrystallized in dichloroethane (100 mL) to give4,4',8-trimethyl-5'-[2-(N-phthalimido)-2-oxa]butylpsoralen (0.68 g,50.8%), as an off-white solid, mp 225°-228° C. ¹ H NMR (CDCl₃): 2.26 (s,3H), 2.46 (s, 3H), 2.51 (d, J=1 Hz, 3H), 3.87 (m, 4H), 4.64 (s, 2H),6.26 (apparent d, J=1 Hz, 1H), 7.42 (s, 1H), 7.64 (multiplet, 4H).

4,4',8-Trimethyl-5'-[4'-(N-phthalimido)-2-oxa]butylpsoralen (1.61 g,3.73 mmol) was stirred with THF (40 mL) and 40 wt % aq methylamine (20mL, 257 mmol) overnight. The solvent was stripped and the residue waspartitioned between dilute aq HCl and dichloromethane. The aqueous layerwas rinsed several more times with dichloromethane then made basic withK₂ CO₃. The base layer was extracted three times with dichloromethane.The combined organic extracts from the base shaken with brine then dried(anhydrous Na₂ SO₄) and stripped to give5'-(4-amino-2-oxa)butyl-4,4',8-trimethylpsoralen (0.71 g, 63.4%), mp126°-129° C. ¹ H NMR (CDCl₃): 2.30 (s, 3H), 2.51 (s, 3H), 2.58 (s, 3H),2.91 (t, J=5 Hz, 2H), 3.59 (t, J=5 Hz, 2H), 4.64 (s, 2H), 6.25 (s, 1H),7.50 (s, 1H).

The above amine (0.71 g, 2.36 mmol) was dissolved in hot ethanol,converted to the acid with 1M HCl in diethylether (3 mL, 3 mmol),decolorized with charcoal, cooled and collected. The solid wasdecolorized again with charcoal and stripped to give5'-(4-amino-2-oxa)butyl-4,4',8-trimethylpsoralen hydrochloride (0.39 g,49.3% yield) as a white solid, mp 235°-236° C. (Note: Other preparationsof this material have given a product with a significantly lower meltingpoint, but identical NMR spectra ). ¹ H NMR (d6-DMSO): 2.32 (s, 3H),2.45 (s, 3H), 2.50 (s, 3H), 3.00 (m, 2H), 3.71 (t, J=5 Hz, 2H), 4.71 (s,2H), 6.33 (s, 1H), 7.79 (s, 1H), 8.15 (br). ¹³ C NMR (d6-DMSO): 7.93,8.57, 19.01, 38.74, 62.66, 66.28, 108.22, 112.42, 113.69, 115.34,116.06, 125.60, 149.38, 150.95, 154.26 (tentatively 2 carbons), 160.26.

EXAMPLE 6 Synthesis of4'-(7-amino-2,5-oxa)heptyl-4,5',8-trimethylpsoralen Hydrochloride(Compound 7)

In this example, the synthesis of Compound 7 is described. The synthesisof 4'-(7-amino-2,5-oxa)heptyl-4,5',8-trimethylpsoralen hydrochlorideproceeds in four (4) steps:

STEP 1:

4'-Chloromethyl-4,5',8-trimethylpsoralen (589 mg, 2.13 mmol), diethyleneglycol (15.4 g, 145 mmol) and acetone (13 mL) were refluxed for 11.5hours. The reaction solution was concentrated to remove acetone and partof the diethylene glycol. To the resulting light brown solution wasadded CHCl₃ (40 mL), then washed with water several times. The CHCl₃layer was dried over anhydrous Na₂ SO₄ and concentrated to give 781 mgof product, 4'-(7-Hydroxy-2,5-oxa)heptyl-4,5',8-trimethylpsoralen,(˜100%). NMR δ2.46 (d, 3H), 2.47 (s, 3H ), 2.51 (s, 3H), 3.58-3.67 (m,8H), 4.67 (s, 2H), 6.18 (s, 1H), 7.57 (s, 1H).

STEP 2:

4'-(7-Hydroxy-2,5-oxa)heptyl-4,5',8-trimethylpsoralen (781 mg, 2.25mmol) was dissolved in CH₂ Cl₂ (2.5 mL) under a N₂ stream at <10° C.Triethylamine (363 mg, 3.59 mmol) was added. Methanesulfonyl chloride(362 mg, 3.16 mmol) was slowly dropped in to keep the temperature below10° C. After addition was completed, the mixture was kept below 10° C.for 15 more minutes. The mixture was stirred at room temperatureovernight then CH₂ Cl₂ (50 mL) was added. The solution was washed withwater (3×60 mL), dried over anhydrous Na₂ SO₄ and concentrated at ≦30°C. Upon vacuum drying, a light brown syrup was obtained[4'-(7-Methanesulfonyloxy-2,5-oxa)heptyl-4,5',8-trimethylpsoralen]; 437mg (76%). NMR δ2.50 (s, 3H), 2.51 (s, 3H), 2.58 (s, 3H), 3.01 (s, 3H),3.66 (m, 4H), 3.77 (t,J=4.6 Hz, 2H), 4.37 (t, J=6 Hz, 2H), 4.69 (s, 2H),6.25 (s, 1H), 7.61 (s, 1H).

STEP 3:

4'-(7-Methanesulfonyloxy-2,5-oxa)heptyl-4,5',8-trimethylpsoralen (288mg, 0.678 mmol) and sodium azide (88.2 mg, 1.36 mmol) were refluxed in 3mL of 95% ethyl alcohol for 8 hours. The reaction solution was let cooland cold water (50 mL) was added. The water layer was poured away. Thecrude material was purified by chromatography on (Silica gel withchloroform eluent) a Chromatotron (Harrison Research, Inc., Palo Alto,Calif.) and vacuum dried to give a light yellow syrup,4'-(7-Azido-2,5-oxa)heptyl-4,5',8-trimethylpsoralen, (123 mg, 49%). NMRδ2.50 (s, 6H), 2.57 (s, 3H), 3.39 (t, J=5.2 Hz, 2H), 3.68 (m, 6H), 4.70(s, 2H), 6.24 (s, 1H), 7.62 (s, 1H).

STEP 4:

4'-(7-Azido-2,5-oxa)heptyl-4,5',8-trimethylpsoralen (122 mg, 0.33 mmol),triphenylphosphine (129 mg, 0.49 mmol) and several drops of water weredissolved in tetrahydrofuran (2 mL). The light yellow clear solution wasstirred at room temperature over a weekend; no starting material wasdetected by TLC. The reaction solution was concentrated and the residuewas dissolved in CHCl₃ (20 mL). The solution was extracted with 0.15Naqueous HCl solution (10 mL then 2×5 mL) and the HCl layers was taken topH 13 by addition of 20% aqueous NaOH solution. The basic solution wasextracted with CHCl₃ (3×15 mL). The combined CHCl₃ layers were washedwith water, dried over anhydrous Na₂ SO₄, concentrated, and vacuum driedto give 63.9 mg of product,4'-(7-amino-2,5-oxa)heptyl-4,5',8-trimethylpsoralen, (56%). TLC showedonly one spot. NMR δ2.50 (s, 3H); 2.50 (s, 3H); 2.57 (s, 3H); 2.86 (t,J=5.3 Hz, 2H); 3.50 (t, J=5.3 Hz, 2H); 3.63 (s, 4H); 4.70 (s, 2H); 6.24(s, 1H); 7.62 (s, 1H). m.p. 170°-173° C.

The solid was dissolved in absolute ethanol, then 1M HCl in ethyl etherwas added, the suspension was filtered and the product rinsed with etherand dried.

EXAMPLE 7 Synthesis of4'-(12-amino-8-aza-2,5-dioxa)dodecyl-4,5',8-trimethylpsoralenDihydrochloride (Compound 8)

The synthesis of4'-(12-amino-8-aza-2,5-dioxa)dodecyl-4,5',8-trimethylpsoralendihydrochloride proceeds in one (1) step from the product of Example 5,method 2, step 2: A solution of4'-(7-methanesulfonyloxy-2,5-oxa)heptyl-4,5',8-trimethylpsoralen (108mg, 0.253 mmol) in 8 mL of acetonitrile was slowly added to a solutionof 1,4-diaminobutane (132 mg, 1.49 mmol) in 2.8 mL of acetonitrile.After refluxing for 8 hours, no starting material remained by TLC. Thereaction mixture was cooled to room temperature and CHCl₃ (25 mL) and 1Naqueous NaOH (25 mL) solution were added. The layers were separated andCHCl₃ (2×10 mL) was used to wash the aqueous layer. Aqueous HCl (0.3N,3×10 mL) was used to extract the product from the combined organicslayers. The HCl layers was treated with 20% aqueous NaOH solution untilpH 13. The combined basic layers were then extracted with CHCl₃ (3×20mL). The CHCl₃ layer was washed with saturated NaCl aqueous solution (10mL) then dried over anhydrous Na₂ SO₄. After concentration and vacuumdrying, 63 mg of product,4'-(12-amino-8-aza-2,5-dioxa)dodecyl-4,5',8-trimethylpsoralendihydrochloride, was obtained (60%). NMR δ1.45 (m, 2H), 2.49 (s, 6H),2.55 (s, 3H), 2.58 (t, 2H), 2.66 (t, J=5.6 Hz, 2H), 2.76 (m, 4H),3.55-3.61 (m, 6H), 4.68 (s, 2H), 6.22 (s, 1H), 7.61 (s, 1H).

EXAMPLE 8 Synthesis of 4'-(2-aminoethyl)-4,5',8-trimethylpsoralenHydrochloride (Compound 3)

The synthesis of 4'-(2-aminoethyl)-4,5',8-trimethylpsoralen proceeds inone (1) step: sodium trifluoroacetoxyborohydride was made by addingtrifluoroacetic acid (296 mg, 2.60 mmol) in 2 mL of THF to a stirredsuspension of sodium borohydride (175 mg, 4.63 mmol) in 2 mL of THF overa period of 10 minutes at room temperature. The resultant suspension wasadded to a suspension of 4'-cyanomethyl-4,5',8-trimethylpsoralen(Kaufman et al., J. Heterocyclic Chem. 19:1051 (1982)) (188 mg, 0.703mmol) in 2 mL of THF. The mixture was stirred overnight at roomtemperature. Several drops of water were added to the reacted lightyellow clear solution to decompose the excess reagent under 10° C. Theresulting mixture was concentrated and 1N aqueous NaOH solution (30 mL)was added. Chloroform (30 mL then 10 mL, 5 mL)) was used to extract theresultant amine. Combined CHCl₃ layers were washed with saturated NaClsolution. The amine was then extracted into aqueous 0.3N HCl (10, 5, 5mL) and the acid layers were taken to pH 13 with 20% aqueous NaOH. CHCl₃(3×10 mL) was used to extract the amine from the combined base layersthen washed with water (2 mL) and dried over anhydrous Na₂ SO₄. Uponconcentration and vacuum drying the amine was obtained as a solid, >95%pure by NMR. NMR δ2.45 (s, 3H); 2.47 (s, 3H); 2.53 (s, 3H); 2.78 (t,J=6.6 Hz, 2H); 3.00 (t, J=6.5 Hz, 2H); 6.20 (s, 1H); 7.44 (s, 1H). Thesolid was dissolved in absolute ethanol. A solution of hydrogen chloridein diethyl ether (1N, 1 mL) was added. The suspension was filtered toobtain compound 3, a light purple solid (32.7 mg, yield 15%), m.p. >237°C. (decomp.)

EXAMPLE 9 4'-(6-Amino-2-aza)hexyl-4,5',8-trimethylpsoralenDihydrochloride (Compound 6)

The synthesis of 4'-(6-amino-2-aza)hexyl-4,5',8-trimethylpsoralendihydrochloride proceeds in one (1) step, as follows: a solution of4'-chloromethyl-4,5',8-trimethylpsoralen (188 mg, 0.68 mmol) in 30 mL ofacetonitrile was added to a solution of 1,4-diaminobutane (120 mg, 1.4mmol) in 7 mL of acetonitrile. After stirring overnight the solvent wasremoved under reduced pressure. Chloroform (10 mL) and 1N NaOH (10 mL)were added to the residue and the mixture was shaken and separated. Theaqueous solution was extracted with a further 2×10 mL of CHCl₃ and thecombined extracts were rinsed with water. The product was then extractedfrom the CHCl₃ solution with 0.3N aqueous HCl and the acidic layer wasthen taken to pH 12 with concentrated NaOH solution. The base suspensionwas extracted with CHCl₃ which was then rinsed with water, dried overNa₂ O₄ and concentrated under reduced pressure to give the amine as thefree base; NMR (CDCl₃); δ1.33 (m, 3H), 1.52 (m, 4H), 2.47 (s, 3H), 2.49(d, J=l.1 nz, 3H), 2.54 (s, 3H), 2.68 (q, J=6.5 Hz, 4H), 3.86 (s, 2H),6.21 (apparent d, J=1.1 Hz, 1H), 7.60 (s, 1H).

The free base, dissolved in about 6 mL of absolute EtOH was treated witha solution of HCl in ether (1.0M, 3 mL). The resultant HCl salt wasfiltered, rinsed with absolute EtOH and dried under vacuum to yield 150mg of compound 6, (55%), m.p. 290° C. (decomposed). Analysis calculatedfor C₁₉ H₂₆ C₁₂ N₂ O₃ •H₂ O: C,54.42; H, 6.73; N, 6.68. Found: C, 54.08;H, 6.45; N, 6.65.

The following compounds were prepared in a similar manner, with thedifferences in synthesis noted:

a) 4'-(4-amino-2-aza)butyl-4,5',8-trimethylpsoralen dihydrochloride(Compound 1), mp 320°-322° C. (decomp). In this synthesis ethylenediamine was used as the diamine.

b) 4'-(5-amino-2-aza)pentyl-4,5',8-trimethylpsoralen dihydrochloride(Compound 5), mp 288° C. (decomp). NMR of free base: d 1.33 (br s, 3H),1.66 (pent, J=6.8 Hz, 2H), 2.47 (s, 3H), 2.50 (d, J=1 Hz, 3H), 2.55 (s,3H), 2.6-2.85 (m, 4H), 3.89 (s, 2H), 6.22 (apparent d, J=1 Hz, 1H), 7.62(s, 1H). For this synthesis, 1,3-diaminopropane was used as the diamine.

c) 4'-(7-amino-2-aza)heptyl-4,5',8-trimethylpsoralen dihydrochloride(Compound 10), mp 300° C. (decomp). NMR of free base: d 1.22 (br s,),1.3-1.6 (m) total 9H, 2.44 (s), 2.50 (s), total 9H, 2.63 (m, 4H), 6.17(s, 1H), 7.56 (s, 1H). Here, 1,5-diaminopentane was used as the diamine.

EXAMPLE 10 5'-(6-Amino-2-aza )hexyl-4,4',8-trimethylpsoralenDihydrochloride (Compound 17)

The synthesis of 5'-(6-amino-2-aza)hexyl-4,4',8-trimethylpsoralendihydrochloride proceeds in one (1) step, as follows: a suspension of5'-chloromethyl-4,4',8-trimethylpsoralen (190 mg, 0.68 mmol) in 30 mL ofacetonitrile was added to a solution of 1,4-diaminobutane (120 mg, 1.4mmol) in 7 mL of acetonitrile. After stirring at room temperatureovernight, the solvent was removed under reduced pressure. Chloroform(10 mL) and 1N NaOH (10 mL) were added to the residue and the mixturewas shaken and separated. The aqueous layer was extracted with a further2×10 mL of CHCl₃ and the combined extracts were rinsed with water. Theproduct was then extracted from the CHCl₃ solution with 0.3N aqueous HCland the acidic layer was then taken to approximately pH 12 withconcentrated NaOH solution. The base suspension was extracted with CHCl₃which was then rinsed with water, dried over Na₂ O₄ and concentratedunder reduced pressure.

The residue was purified by column chromatography on silica gel withCHCl₃ :EtOH:Et₃ N (9:1:0.25). The fractions containing the product werecombined and stripped of the solvent to give the free amine. NMR(CDCl₃): δ1.35 (m, 3H); 1.49 (m, 4H); 2.22 (s, 3H); 2.46 (d, J=1.1 Hz,3H); 2.51 (S, 3H); 2.65 (m, 4H); 3.88 (s, 2H); 6.17 (apparent d, 1 Hz);7.40 (s, 1H).

The free base, dissolved in absolute EtOH (˜6 mL) was treated with asolution of HCl in ether (1.0M,˜3 mL). The resultant HCl salt wasfiltered, rinsed with absolute EtOH and dried under vacuum to yield 100mg (36.3%) of product, 5'-(6-Amino-2-aza)hexyl-4,4',8-trimethylpsoralendihydrochloride, m.p. 288° C. (decomposed).

5'-(4-Amino-2-aza)butyl-4,4',8-trimethylpsoralen dihydrochloride(Compound 16) was prepared in the same manner, except that ethylenediamine was used as the diamine. NMR of free base: δ1.83 (br s, 3H),2.27 (s, 3H), 2.51 (s, 3H), 2.58 (s, 3H), 2.74 (m, 2H), 2.87 (m, 2H),3.95 (s, 2H), 6.24 (s, 1H), 7.46 (s, 1H).

EXAMPLE 114'-(14-Amino-2,6,11-triaza)tetradecyl-4,5',8-trimethylpsoralenTetrahydrochloride (Compound 15)

The synthesis of4'-(14-amino-2,6,11-triaza)tetradecyl-4,5',8-trimethylpsoralentetrahydrochloride proceeds in one (1) step, as follows. To a solutionof 0.5 g (2.5 mmol) of spermine (Aldrich, Milwaukee, Wis.) in 10 ml ofmethanol was added a 5N methanolic solution of HCl (concentrated HCldiluted with MeOH to 5N) to adjust to pH 5-6, followed by 0.128 g (0.5mmol) of 4,5',8-trimethylpsoralen-4'carboxaldehyde, 20 mg (0.3 mmol) ofNaBH₃ CN and 3 mL of MeOH. The reaction mixture was stirred at roomtemperature overnight. A solution of 5N methanolic HCl was added untilpH<2 and methanol was removed under reduced pressure. The residue wastaken up in about 100 mL of water and rinsed with three 25 mL portionsof CHCl₃. The aqueous solution was brought to pH>10 with concentratedNaOH and extracted with three 25 mL portions of CHCl₃. These finalextracts were combined and washed with water, dried (Na₂ O₄) andevaporated to give the free base of the amine, ≧95% pure by NMR. NMR(CDCl₃): d 1.31 (m, 5H), 1.45 (pent, J=3.41 Hz, 4H), 1.65 (m, 4H), 2.46(s, 3H), 2.49 (d, J=1.14 Hz, 3H), 2.66 (m, 15H), 3.85 (s, 2H), 6.21 (s,1H)m 7.60 (s, 1H).

The free amine was dissolved in absolute ethanol and HCl (anhydrous, 1Nin ethyl ether) was added. The hydrochloride salt was filtered andwashed with absolute ethanol and dried under vacuum at room temperaturegiving 80.2 mg of product,4'-(14-amino-2,6,11-triaza)tetradecyl-4,5',8-trimethylpsoralentetrahydrochloride, as a light yellow solid.

EXAMPLE 12

An r-17 bacteriophage assay was used in this example to predict pathogeninactivation efficiency and to determine nucleic acid binding of thephotoreactive binding compounds of the present invention. In the r-17assay, the bacteriophage was placed in a solution with each compoundtested and was then irradiated. The ability of the phage to subsequentlyinfect bacteria and inhibit their growth was measured. The bacteriophagewas selected for its relatively accessible nucleic acid such that theculture growth inhibition would accurately reflect nucleic acid damageby the test compounds. The bacteriophage assay for nucleic acid bindingto test compounds offers a safe and inexpensive procedure to identifycompounds likely to display efficient pathogen inactivation. Previousexperiments support that the r-17 assay accurately measures HIV-1sensitivity to similar compounds.

The r-17 was grown up in Hfr 3000 bacteria, approximate titer 5×10¹¹(R17 and Hfr 3000 were obtained from American Tissue Culture Collection(ATCC), Washington, D.C.) The R17 phage stock was added to a solution of15% fetal bovine serum in Dulbecco's Modified Eagles Medium (DMEM) to afinal phage concentration of 10⁹ /mL. An aliquot (0.5 mL) wastransferred to a 1.5 mL snap-top polyethylene tube. An aliquot(0.004-0.040 mL) of the test compound stock solution prepared in water,ethanol or dimethylsulfoxide at 0.80-8.0 mM was added to the tube.Compounds were tested at concentrations between 4 μM and 320 μM. (AMT iscommercially available from HRI, Inc., Concord, Calif.; 8-MOP iscommercially available from Sigma, St. Louis, Mo.). The tubes wereplaced in a light device as described in EXAMPLE 1 and irradiated forbetween 1 and 10 minutes. Sterile 13 mL dilution tubes were prepared;each test compound required one tube with 0.4 mL of Luria broth (LB) andfive tubes containing 0.5 mL of LB broth. To make the dilutions, a 0.100mL aliquot of the irradiated solution of phage and test compound wasadded to the first dilution tube of 0.4 mL of media then 0.020 mL ofthis solution was added to the second tube of 0.5 mL medium (1:25). Thesecond solution was then diluted serially (1:25) into the remainingtubes. To each diluted sample was added 0.050 mL of Hfr 3000 bacteriacultured overnight and 3 mL of molten LB top agar and the mixedmaterials were poured onto LB broth plates. After the top agar hardened,the plates were incubated at 37° C. overnight. The plaque forming unitswere then counted the following morning and the titer of the phageremaining after phototreatment was calculated based on the dilutionfactors.

The following controls were run: the "phage only" in which phage was nottreated with test compound and not irradiated (listed as "startingtiter" in the tables below); the "UV only" in which the phage wasirradiated in the absence of test compound; and the "dark" control inwhich the phage/test compound solution was not irradiated before it wasdiluted and plated.

TABLE 5, below, shows three different experiments which tested Compound1 according to the R17 protocol just described. A comparison of valuesfor the control samples in runs 1-3 (values in bold) shows that neitherthe "UV only" nor the "dark" controls result in significant bacterialkill (at most, 0.3 logs killed in the "UV only" control and 0.1 logskilled in the "dark" control).

The "UV only" control was repeated in many similar experiments withother compounds of the present invention and consistently showed nosignificant kill. (Data not shown). Thus, the "UV only" control is notshown in the tables and figures that follow, although it was performedin every experiment in this example. As for the "dark" control, aftermany trials with various compounds of the present invention, it becameapparent that regardless of the type of substitution on the 4' positionof the psoralen, no experimentally significant bacterial inactivationwas observed in the dark. (Data not shown). For example, in Table 5,experiment 1 shows 0.1 logs kill with compound 1 in the dark. Incontrast, when Compound 1 is irradiated for just 1 minute, the resultingdrop in titer is >6.7 logs. Therefore, "dark" controls were not run forthe later tested compounds and where run, are not shown in the tablesand figures that follow.

                  TABLE 5                                                         ______________________________________                                        EXPERIMENT                         LOGS                                       #           TREATMENT   LOG TITER  KILLED                                     ______________________________________                                        1           phage only  7.7        --                                                     uva only (10')                                                                            7.4        0.3                                                    compound only                                                                             7.6        0.1                                                    (32 μM)                                                                    32 μM cmpd                                                                             <1         >6.7                                                   1' uva                                                                        32 μM cmpd                                                                             <1         >6.7                                                   10' uva                                                           2           phage only  7.8        --                                                     uva only (10')                                                                            7.6        0.2                                                    compound only                                                                             7.7        0.1                                                    (3.2 μM)                                                                   3.2 μM cmpd                                                                            6.9        0.9                                                    1' uva                                                                        3.2 μM cmpd                                                                            6.1        1.7                                                    10' uva                                                           3           phage only  7.3        --                                                     uva only (1')                                                                             7.3        0                                                      compound only                                                                             7.3        0                                                      (16 μM)                                                                    4 μM cmpd                                                                              6.3        1.0                                                    1' uva                                                                        8 μM cmpd                                                                              5.6        1.7                                                    1' uva                                                                        16 μM cmpd                                                                             3.9        3.4                                                    1' uva                                                            ______________________________________                                    

Tables 6-9, below, and FIGS. 11-13 show the results of the R17 assay forseveral of the 4'-primaryamino-substituted psoralen compounds of thepresent invention. The data in Tables 7 and 8 appears in FIGS. 11 and12, respectively. 5'-Primaryamino-substituted psoralen compounds of thepresent invention, which have substitutions on the 5' position similarto the 4'-primaryamino-substituted psoralen compounds, were also testedat varying concentration, as described above in this example, and areshown to exhibit comparable inactivation efficiency. The results forthese compounds are shown in FIGS. 14 and 15, below.

The compounds of the present invention having substitutions on the 4'position of the psoralen ring proved to be active in killing R17, asshown in

                  TABLE 6                                                         ______________________________________                                        Starting titer of R17: approx. 7.5 logs                                       1 Minute Irradiation                                                                       R17 log kill                                                            Cmpd. (32 μM)                                                       ______________________________________                                               AMT   >6.7                                                                    8-MOP 0                                                                       1     >6.6                                                             ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Starting titer approx. 7.2 logs R17                                           1 minute irradiation                                                                   R17 log kill                                                         Compound   8 μM      16 μM                                                                              32 μM                                      ______________________________________                                        AMT        2.7          4.6     >6.2                                          1          1.7          2.8     5.3                                           2          3.8          >6.2    >6.2                                          3          >6.2         >6.2    >6.2                                          ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Starting titer approx. 7.1 logs                                               1 minute irradiation = 1.2 J/cm.sup.2                                                 R17 log kill                                                          Cmpd.     8 μM 16 μM   32 μM                                                                            64 μM                                   ______________________________________                                        AMT       --      4.5        4.8   --                                         3         5.6     >6.1       --    --                                         4         --      2.3        4.3   >6.1                                       5         --      5.6        >6.1  >6.1                                       6         --      >6.1       >6.1  >6.1                                       ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Starting titer approx. 7.1 logs R17.                                          1 Minute Irradiation.                                                                 R17 log kill                                                          Cmpd.     8 μM 16 μM   32 μM                                                                            64 μM                                   ______________________________________                                        AMT       --      >6         >6    --                                         6         >6      >6         --    --                                         7         --      >6         >6    >6                                         ______________________________________                                    

the tables above. In Table 7, it is apparent that compound 1 of thepresent invention exhibits much higher R17 inactivation efficiency thandoes 8-MOP. As shown in Table 7 and FIG. 11, Compound 1 is one of theless active compounds of the present invention. Both Compounds 2 and 3show higher log inactivation than Compound 1 at each concentrationpoint. These results support that the compounds of the present inventionare generally much more active than 8-MOP.

The compounds of the present invention also have similar or better R17inactivation efficiency than AMT. In Tables 7 and 8, and FIGS. 11-15,all compounds of the present invention achieve R17 log inactivation atlevels comparable to AMT. Compounds 2 and 3 (Table 6, FIG. 11),Compounds 5 and 6 (Table 8, FIG. 12), and Compound 16 (FIG. 15) exhibitsignificantly higher inactivation efficiency than does AMT.

Compounds of the present invention were also tested at a constantconcentration for varying doses of UV light. Three sets of 1.5 mL tubeswere prepared containing 0.6 mL aliquots of R17 in DMEM (prepared asdescribed above). The compound tested was added at the desiredconcentration and the samples were vortexed. The samples were thenirradiated at intervals of 1.0 J/cm², until 3.0 J/cm² was reached.Between each 1.0 J/cm² interval, 1001 μL was removed from each sampleand placed in the first corresponding dilution tube, then fivesequential dilutions were performed for each compound tested, at all 3irradiation doses, as described above in this example.

Then 50 μL of Hfr 3000 bacteria was added to each tube, 3 mL of top agarwas added and the tube contents were vortexed. The contents of each tubewas poured into its own LB plate and the plates were incubated overnightat 37° C. Plaques were counted by visual inspection the followingmorning.

The results of the assay for several 4' and 5'-primaryamino-substitutedpsoralen compounds are shown in FIGS. 16-22. This data further supportsthat the compounds of the present invention are comparable to AMT intheir ability to inactivate R17. Further, Compounds 6 (FIG. 16), 10(FIG. 17), 12 (FIG. 18), 15 (FIG. 19 and 22), and Compound 17 (FIG. 20),all were more efficient at inactivating R17 than was AMT.

EXAMPLE 13

Pathogen inactivation efficiency of several compounds of the presentinvention was evaluated by examining the ability of the compounds toinactivate cell-free virus (HIV). Inactivation of cell-free HIV wasperformed as follows.

As in the R17 assay, small aliquots of the compounds listed in TABLES 10and 11, below, at the concentrations listed in the table, were added tostock HIV-1 to a total of 0.5 mL. The stock HIV (10⁵ -10⁷ plaque formingunits/mL) was in DMEM/15% FBS. The 0.5 mL test aliquots were placed in24 well polystyrene tissue culture plates and irradiated with 320-400 nm(20 mW/cm²) for 1 min on a device similar to the device of Example 1.The photoactivation device used here was previously tested and found toresult in light exposure comparable to the Device of Example 1. (Datanot shown). Controls included HIV-1 stock only, HIV-1 plus UVA only, andHIV-1 plus the highest concentration of each psoralen tested, with noUVA. Post irradiation, all samples were stores frozen at -70° C. untilassayed for infectivity by a microtiter plaque assay. Aliquots formeasurement of residual HIV infectivity in the samples treated with acompound of the present invention were withdrawn and cultured.

Residual HIV infectivity was assayed using an MT-2 infectivity assay.(Previously described in Hanson, C. V., Crowford-Miksza, L. andSheppard, H. W., J. Clin. Micro 28:2030 (1990)). The assay medium was85% DMEM (with a high glucose concentration) containing 100 μg ofstreptomycin, 100 U of penicillin, 50 μg of gentamicin, and 1 μg ofamphotericin B per mL, 15% FBS and 2 μg of Polybrene (Sigma ChemicalCo., St. Louis, Mo.) per mL. Test and control samples from theinactivation procedure were diluted in 50% assay medium and 50% normalhuman pooled plasma. The samples were serially diluted directly in96-well plates (Corning Glass Works, Corning, N.Y.). The plates weremixed on an oscillatory shaker for 30 seconds and incubated at 37° C. ina 5% CO₂ atmosphere for 1 to 18 hours. MT-2 cells (0.025 mL) [clonealpha-4, available (catalog number 237) from the National Institutes ofHealth AIDS Research and Reference Reagent Program, Rockville, Md.] wereadded to each well to give a concentration of 80,000 cells per well.After an additional 1 hour of incubation at 37° C. in 5% CO₂, 0.075 mLof assay medium containing 1.6% SeaPlaque agarose (FMC Bioproducts,Rockland, Me.) and prewarmed to 38.5° C. was added to each well. Theplates were kept at 37° C. for a few minutes until several plates hadaccumulated and then centrifuged in plate carriers at 600×g for 20minutes in a centrifuge precooled to 10° C. In the centrifuge, cellmonolayers formed prior to gelling of the agarose layer. The plates wereincubated for 5 days at 37° C. in 5% CO₂ and stained by the addition of0.05 mL of 50 μg/mL propidium iodide (Sigma Chemical Co.) inphosphate-buffered saline (pH 7.4) to each well. After 24 to 48 hours,the red fluorescence-stained microplaques were visualized by placing theplates on an 8,000 μW/cm² 304 nm UV light box (Fotodyne, Inc., NewBerlin, Wis.). The plaques were counted at a magnification of ×20 to ×25through a stereomicroscope. The results are shown in TABLES 10 and 11,below. "n" represents the number of runs for which the data point is anaverage.

The results support that the compounds of the present invention areeffective in inactivating HIV. In fact, the data for concentrations of64 μM of compound or higher suggests that compounds 2 and 3 aresignificantly more active than AMT, which was previously thought to beone of the most active anti-viral psoralens. At lower concentrations,Compound 6 is able to kill a higher log of HIV (3.1 logs at 32 μM) thanis AMT (2.5 logs at 32 μM). The other compounds listed in TABLE 9display inactivation efficiency in the same range as AMT.

                  TABLE 10                                                        ______________________________________                                        1 minute irradiation                                                          HIV starting titer: approximately 5 logs                                              HIV log kill                                                          COMPOUND  16 μM 32 μM  64 μM                                                                              128 μM                                ______________________________________                                        AMT       1.4      1.9->3.6  3.9->3.6                                                                              >4.1                                     1         --       --        2.1     >2.8                                     2         1.4      3.8       >4.5    >4.5                                     3         --       2.7       >3.8    >3.8                                     4         --       2.2       >3.6    >3.6                                     5         0.9      1.3       >2.6    --                                       6         2.0      3.1       >3.8    --                                       7         0.8      2.1       3.5     --                                       8         1.1      1.9       3.7     >3.7                                     ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                        HIV starting titer: approximately 5.4 logs                                    1 minute irradiation                                                                  HIV log kill                                                          COMPOUND     16 μM    32 μM                                                                              64 μM                                     ______________________________________                                         6           2.1         3.2     >2.8                                          9           0.8         1.4     2.7                                          10           2.0         >3.5    >3.5                                         12           0.4         0.8     1.3                                          17           1.2         2.9     3.4                                          18           1.0         1.0     3.1                                          ______________________________________                                    

EXAMPLE 14

This example describes the protocol for inactivation of Duck Hepatitis BVirus (DHBV), a model for Hepatitis B Virus, using compounds of thepresent invention.

DHBV in duck yolk was added to platelet concentrate (PC) to a finalconcentration of 2×10⁷ particles per mL and mixed by gentle rocking for≧15 min. Psoralens S-70, S-59 and AMT were added to 3 mL aliquots of PCin a Teflon™ mini-bag at concentrations of 35, 70, and 100 mM. Samples,including controls without added psoralen, were irradiated with 5 J/CM²UVA, with mixing at 1 J/cm² increments. After irradiation, leukocytesand platelets were separated from virus by centrifugation. Thesupernatant containing DHBV was digested overnight with 50 μg/mLproteinase K in a buffer containing 0.5% sodium dodecyl sulphate, 20 mMTris buffer, pH 8.0, and 5 mM EDTA at 55° C. Samples were extracted withphenol-chloroform and chloroform, followed by ethanol precipitation.Purified DNA was then used in PCR amplification reactions with astarting input of 10⁶ DHBV genomes from each sample. PCR amplicons weregenerated using primers pairs DCD03/DCD05 (127 bp), DCD03/DCD06 (327 bp)and DCD03/DCD07 (1072 bp). PCR was performed in a standard PCR buffercontaining 0.2 mM each deoxyribonucleoside 5'-triphosphates (dATP, dGTP,dCTP, and dTTP), 0.5 mM each primer, and 0.5 units Taq polymerase per100 ml reaction. 30 cycles of amplification were performed with thefollowing thermal profile: 95° C. 30 sec, 60° C. 30 sec, 72° C. 1 min.The amplification was followed by a 7 min incubation at 72° C. to yieldfull length products. [lambda-³² P] dCTP was added at an amount of 10mCi per 100 ml in order to detect and quantify the resulting products.Products were separated by electrophoresis on denaturing polyacrylamideslab gels and counted. The absence of signal in a given reaction wastaken to indicate effective inactivation of DHBV.

The results showed that the smaller amplicons displayed increasinginactivation as a function of psoralen concentration for all psoralenstested. At the same concentrations, S-59 and S-70 inhibited PCR of thesmaller amplicons better than did AMT. For the 1072 bp amplicon,complete inhibition of PCR was observed at all concentrations of S-59and S-70, whereas the sample without psoralen gave a strong signal. AMTinhibited PCR amplification of the 1072 bp amplicon at the 70 and 100 mMlevels, but a signal could be detected when AMT was used at 35 mM finalconcentration.

EXAMPLE 15

In Example 13, the compounds of the present invention were tested fortheir ability to inactivate virus in DMEM/15% FBS. In this example, thecompounds are tested in both 100% plasma and predominantly syntheticmedia, to show that the methods of the present invention are notrestricted to any particular type of medium.

For the samples in synthetic media: standard human platelet concentrateswere centrifuged to separate plasma. Eighty-five percent of the plasmawas then expressed off and replaced with a synthetic medium (referred toas "Sterilyte™ 3.0") containing 20 mM Na acetate, 2 mM glucose, 4 mMKCl, 100 mM NaCl, 10 mM Na₃ Citrate, 20 mM NaH₂ PO₄ /Na₂ HPO₄, and 2 mMMgCl₂. H9 cells infected with HIV were added to either the 85%Sterilyte™ 3.0 platelet concentrates or standard human plateletconcentrates (2.5×10⁷ cells per concentrate), final concentration 5×10⁵cells/mL. The platelet concentrates were placed in Teflon™ modified FL20or Teflon™ Minibags (American Fluoroseal Co., Silver Springs, Md.),treated with one of the compounds shown in FIGS. 23 and 24, at theconcentrations shown, and then irradiated with 320-400 nm (20 mW/cm²)for 5 J/cm² (for plasma samples) or 2 J/cm² (for 85% Sterilyte™ 3.0samples) on a device similar to the Device of Example 1. Thephotoactivation device used here was previously tested and found toresult in light exposure comparable to the Device of Example 1. (Datanot shown). Aliquots for measurement of residual HIV infectivity in thesamples treated with a compound of the present invention were withdrawnand cultured.

For samples run in plasma: H9 cells infected with HIV were added tostandard human platelet concentrates (2.5×10⁷ cells per concentrate),final concentration 5×10⁵ cells/mL. Aliquots of HIV contaminatedplatelet concentrate (5 mL) were placed in water jacketed Pyrexchambers. The chambers had previously been coated on the inside withsilicon. The platelet concentrates were treated with one of thecompounds listed in TABLES 10 and 11, below, at the concentrationslisted in the table, and then irradiated with 320-400 nm (20 mW/cm²) for1 minute on a device similar to the Device of Example 1. Thephotoactivation device used here was previously tested and found toresult in light exposure comparable to the Device of Example 1. (Datanot shown). Aliquots for measurement of residual HIV infectivity in thesamples treated with a compound of the present invention were withdrawnand cultured. Residual HIV infectivity was assayed for both the plasmaand the 85% Sterilyte™ samples using an MT-2 infectivity assay.(Detailed in Example 13, above, and previously described in Hanson, C.V., et al., J. Clin. Micro 28:2030 (1990)). The results are shown inFIGS. 23 and 24.

The results support that the compounds of the present invention areeffective in inactivating HIV in both plasma and synthetic medium.Comparing FIGS. 18 and 19, the inactivation curves appear to be thesame, both achieving approximately 5 logs of inactivation at 64 μMconcentrations of compound. However, the inactivation in synthetic mediawas performed with only 2 J/cm² irradiation, 3 J/cm² less than thatrequired to acheive the same inactivation in plasma. Thus, it appearsfrom the data that synthetic media facilitates the inactivation methodsof the present invention.

EXAMPLE 16

In this example bacterial inactivation by the photoreactive nucleic acidbinding compounds of the present invention was measured as a function ofthe ability of the bacteria to subsequently replicate. A gram negativebacteria was chosen as representative of the more difficult bacterialstrains to inactivate.

The bacteria, a strain of Pseudomonus, was innoculated into LB with asterile loop and grown overnight in a shaker at 37° C. Based on theapproximation that one OD at 610 nm is equivalent to 5×10⁸ colonyforming units (cfu)/mL, a 1:10 dilution of the culture was measured on aspectrophotometer, (manufactured by Shimatsu). The bacterial culture wasadded to a solution of 15% fetal bovine serum in DMEM to a finalbacteria concentration of approximately 10⁶ /mL. An aliquot (0.8 mL) wastransferred to a 1.5 mL snap-top polyethylene tube. An aliquot(0.004-0.040 mL) of the test compound stock solution prepared in water,ethanol or dimethylsulfoxide at 0.80-8.0 mM was added to the tube.Compounds were tested at a concentration of 16 μM. The tubes were placedin a light device as described in EXAMPLE 1 and irradiated with 1.3J/cm², 1.2 J/ cm², and finally 2.5 J/cm², for a total of 5 J/cm². 150 μLwere removed for testing after each pulse period. Sterile 13 mL dilutiontubes were prepared; each test compound required one tube with 0.4 mL ofLB broth and four tubes containing 0.5 mL of LB broth. To make thedilutions, a 0.050 mL aliquot of the irradiated solution of phage andtest compound was added to the first dilution tube of 0.5 mL of mediathen 0.050 mL of this solution was added to the second tube of 0.5 mLmedium (1:10). The second solution was then diluted serially (1:10) intothe remaining tubes. 100 μL of the original sample and each dilution areplated separately onto LB agar plates and incubated at 37° C. overnight.The colony forming units were then counted the following morning and thetiter of the phage remaining after phototreatment was calculated basedon the dilution factors.

The following controls were run: the "bacteria only" in which bacteriawas not treated with test compound and not irradiated (listed as"starting titer" in the tables below); the "UV only" in which thebacteria was irradiated in the absence of test compound. Dark controlswere not performed here for reasons set forth in Example 12 above.

The results were as follows. The starting titer of bacteria was 6.5logs. After 5 J/cm² irradiation, the log kill for the various compoundstested were as follows: 8-MOP-1.9 logs, AMT-5.2 logs, Compound 2->5.5,Compound 6->5.5. From these results, it is clear that the compounds ofthe present invention are more efficient than both AMT and 8-MOP atinactivating a gram negative bacteria.

EXAMPLE 17

In the above examples, psoralens of the present invention have beendemonstrated to be effective for inactivating pathogens, such asbacteria (pseudomonus), bacteriophage (R17) and viruses (HIV and DHBV).Without intending to be limited to any method by which the compounds ofthe present invention inactivate pathogens, it is believed thatinactivation results from light induced binding of the psoralens to thenucleic acid of the pathogens. As discussed above, AMT is known both forits pathogen inactivation efficiency and its accompanying mutagenicaction in the dark at low concentrations. In contrast, the less activepsoralens, such as 8-MOP, that have been examined previously, showsignificantly less mutagenicity. This example establishes thatphotobinding and mutagenicity are not linked phenomenon in the compoundsof the present invention. The psoralens of the present invention haveexceptional pathogen inactivation efficiency while displaying onlyminimal mutagenicity.

In this example the compounds of the present invention are tested fortheir dark mutagenicity using an Ames assay. The procedures used for theSalmonella mutagenicity test as described in detail by Maron and Ameswere followed exactly. Maron, D. M. and B. N. Ames, Mutation Research113:173 (1983). A brief description for each procedure is given here.The tester strains TA97a, TA98, TA100, TA102, TA1537 and TA1538 wereobtained from Dr. Ames. TA97a, TA98, TA1537 and TA1538 are frameshifttester strains. TA100 and TA102 are base-substitution tester strains.Upon receipt each strain was cultured under a variety of conditions toconfirm the genotypes specific to the strains.

The standard Salmonella tester strains used in this study requirehistidine for growth since each tester strain contains a different typeof mutation in the histidine operon. In addition to the histidinemutation, these tester strains contain other mutations, described below,that greatly increase their ability to detect mutagen.

Histidine Dependence:

The requirement for histidine was tested by streaking each strain firston a minimal glucose plate supplemented only with biotin and then on aminimal glucose plate supplemented with biotin and histidine. Allstrains grew the lack of growth of the strains in the absence ofhistidine.

rfa Mutation:

A mutation which causes partial loss of the lipopolysaccharide barrierthat coats the surface of the bacteria thus increasing permeability tolarge molecules was confirmed by exposing a streaked nutrient agar platecoated with the tester strain to crystal violet. First 100 μL of eachculture was added to 2 mL of molten minimal top agar and poured onto anutrient agar plate. Then a sterile filter paper disc saturated withcrystal violet was placed at the center of each plate. After 16 hours ofincubation at 37° C. the plates were scored and a clear zone of nobacterial growth was found around the disc, confirming the rfa mutation.

uvrB Mutation:

Three strains used in this study contain a deficient UV repair system(TA97a, TA98, TA100, TA1537 and TA1538). This trait was tested for bystreaking the strains on a nutrient agar plate, covering half of theplate, and irradiating the exposed side of the plate with germicidallamps. After incubation growth was only seen on the side of the plateshielded from UV irradiation.

R-factor:

The tester strains (TA97a, TA98, TA100, and TA102) contain the pKM101plasmid that increases their sensitivity to mutagens. The plasmid alsoconfers resistance to ampicillin to the bacteria. This was confirmed bygrowing the strains in the presence of ampicillin.

pAQ1:

Strain TA102 also contains the pAQ1 plasmid that further enhances itssensitivity to mutagens. This plasmid also codes for tetracyclineresistance. To test for the presence fo this plasmid TA102 was streakedon a minimal glucose plate containing histidine, biotin, andtetracycline. The plate was incubated for 16 hours at 37° C. The strainshowed normal growth indicating the presence of the pAQ1 plasmid.

The same cultures used for the genotype testing were again cultured andaliquots were frozen under controlled conditions. The cultures wereagain tested for genotype to confirm the fidelity of the genotype uponmanipulation in preparing the frozen permanents.

The first tests done with the strains were to determine the range ofspontaneous reversion for each of the strains. With each mutagenicityexperiment the spontaneous reversion of the tester strains to histidineindependence was measured and expressed as the number of spontaneousrevertants per plate. This served as the background controls. A positivemutagenesis control was included for each tester strain by using adiagnostic mutagen suitable for that strain (2-aminofluorene at 5mg/plate for TA98 and sodium azide at 1.5 mg/plate for TA100).

For all experiments, the pre-incubation procedure was used. In thisprocedure one vial of each tester strain was thawed and 20 μL of thisculture was added to 6 mL of Oxoid Nutrient Broth #2. This solution wasallowed to shake for 10 hours at 37° C. In the pre-incubation procedure,0.1 mL of this overnight culture was added to each of the requirednumber of sterile test tubes. To half of the tubes 0.5 mL of a 10% S-9solution containing Aroclor 1254 induced rat liver extract (MolecularToxicology Inc., Annapolis, Md.), and MgCl₂, KCl, glucose-6-phosphate,NADP, and sodium phosphate buffer (Sigma, St. Louis, Mo.) were added. Tothe other half of the tubes 0.5 mL of 0.2M sodium phospate buffer, pH7.4, was used in place of the S-9 mixture (the S-9 samples). Finally 0.1mL of the test solution containing either 0, 0.1, 0.5, 1, 5, 10, 50,100, 250, or 500 μg/mL of the test compound was added. The 0.7 mLmixture was vortexed and then pre-incubated while shaking for 20 minutesat 37° C. After shaking, 2 mL of molten top agar supplemented withhistidine and biotin were added to the 0.7 mL mixture and immediatelypoured onto a minimal glucose agar plate (volume of base agar was 20mL). The top agar was allowed 30 minutes to solidify and then the plateswere inverted and incubated for 44 hours at 37° C. After incubation thenumber of revertant colonies on each plate were counted. The resultsappear in TABLES 12(A)-18(B), below. ("n" represents the number ofreplicates performed for each data point.)

                                      TABLE 12 (A)                                __________________________________________________________________________    AMT                                                                                TA97a -                                                                            TA97a +                                                                             TA98 -                                                                             TA98 +                                                                              TA100 -                                                                             TA100 +                                      STRAIN                                                                             S9   S9    S9   S9    S9    S9                                           __________________________________________________________________________    Dose                                                                          μg/plate                                                                   0    109  158   20    25   126   123                                                n = 23                                                                             n = 39                                                                              n = 38                                                                             n = 53                                                                              n = 41                                                                              n = 56                                      0.1  14   -23    3     1   -10   -16                                               n = 3                                                                              n = 6 n = 3                                                                              n = 6 n = 3 n = 6                                        0.5   9    32    5     3   13    -12                                               n = 3                                                                              n = 6 n = 3                                                                              n = 6 n = 3 n = 6                                        1    54    32    5    21   17    -19                                               n = 3                                                                              n = 6 n = 3                                                                              n = 6 n = 3 n = 6                                        5    73   149   16   232   59    -6                                                n = 3                                                                              n = 6 n = 6                                                                              n = 9 n = 9  n = 12                                      10              20   403   105   17                                                           n = 9                                                                              n = 9  n = 15                                                                              n = 15                                      50              69   620   73    52                                                           n = 9                                                                              n = 9 n = 9 n = 9                                        100             114  745   75    85                                                           n = 9                                                                              n = 9 n = 9 n = 9                                        250             112  933   24    89                                                           n = 6                                                                              n = 6 n = 6 n = 6                                        Positive  5 μg/plate                                                                            5 μg/plate                                                                       1.5 μg/plt                                      Control   2-Amino    2-Amino                                                                             sodium                                                       fluorene   fluorene                                                                            azide                                                        808        1154  965                                                           n = 21     n = 35                                                                              n = 38                                            __________________________________________________________________________

                                      TABLE 12 (B)                                __________________________________________________________________________    AMT                                                                                TA102 -                                                                             TA102 +                                                                            TA1537 -                                                                            TA1537 +                                                                            TA1538 -                                                                            TA1538 +                                    STRAIN                                                                             S9    S9   S9    S9    S9    S9                                          __________________________________________________________________________    Dose                                                                          μg/plate                                                                   0    346    404   9     9   15    19                                                n = 26                                                                              n = 41                                                                             n = 30                                                                             n = 45                                                                               n = 30                                                                             n = 42                                      0.1   27   -20    0    2    3      3                                               n = 3 n = 6                                                                              n = 3 n = 6 n = 3 n = 6                                       0.5   47      5   3    2    4     13                                               n = 3 n = 6                                                                              n = 9 n = 12                                                                              n = 9 n = 12                                      1     88   -17    5    3    4     37                                               n = 3 n = 6                                                                              n = 9 n = 12                                                                              n = 9 n = 12                                      5    266     51  44   22    13    177                                              n = 3 n = 6                                                                              n = 9 n = 12                                                                               n = 18                                                                             n = 21                                      10               52   30    14    255                                                         n = 9 n = 9 n = 9 n = 9                                       50              2688  94                                                                      n = 9 n = 9                                                   100             2058  686                                                                     n = 9 n = 9                                                   250             434   3738                                                                    n = 9 n = 12                                                  Positive                                                                           100 μg/pl                                                                             10 μg/plt                                                                        10 μg/plt                                                                              5 μg/plate                               Control                                                                            hydrogen   9-Amino                                                                             2-Amino     2-Amino                                          peroxide   acridine                                                                            fluorene    fluorene                                         660        284   73          1064                                             n = 23     n = 6 n = 24      n = 30                                      __________________________________________________________________________

                  TABLE 13 (A)                                                    ______________________________________                                        8-MOP                                                                                 TA102 -   TA102 +   TA1537 -                                                                              TA1537 +                                  STRAIN  S9        S9        S9      S9                                        ______________________________________                                        Dose                                                                          μg/plate                                                                   0       346        404      9       9                                                 n = 26    n = 41    n = 30  n = 45                                    1       -55       -46                                                                 n = 14    n = 17                                                      10      -57       -27                                                                 n = 14    n = 17                                                      30                          5       1                                                                     n = 3   n = 6                                     60                          3       1                                                                     n = 3   n = 6                                     90                          -1      -4                                                                    n = 3   n = 6                                     100     217        290                                                                n = 14    n = 17                                                      500     781       1179                                                                n = 11    n = 11                                                      Positive                                                                              100                 10 μg/plt                                                                          10 μg/plt                              Control μg/plt           9-Amino-                                                                              2-Amino-                                          hydrogen            Acridine                                                                              fluorene                                          peroxide            284     73                                                660                 n = 6   n = 24                                            n = 23                                                                ______________________________________                                    

                  TABLE 13 (B)                                                    ______________________________________                                        8-MOP                                                                                 TA102 -   TA102 +   TA1537 -                                                                              TA1537 +                                  STRAIN  S9        S9        S9      S9                                        ______________________________________                                        Dose                                                                          μg/plate                                                                   0       346       404       9       9                                                 n = 26    n = 41     n = 30  n = 45                                   1       -55       -46                                                                 n = 14    n = 17                                                      10      -57       -27                                                                 n = 14    n = 17                                                      30                          5       1                                                                     n = 3   n = 6                                     60                          3       1                                                                     n = 3   n = 6                                     90                          -1      -4                                                                    n = 3   n = 6                                     100     217        290                                                                n = 14    n = 17                                                      500     781       1179                                                                n = 11    n = 11                                                      Positive                                                                              100 μg/plt       10 μg/plt                                                                          10 μg/plt                              Control hydrogen            9-Amino-                                                                              2-Amino-                                          peroxide            Acridine                                                                              fluorene                                          660                 284     73                                                n = 23              n = 6   n = 24                                    ______________________________________                                    

                  TABLE 14                                                        ______________________________________                                        Compound 1                                                                            TA100 -    TA100 +   TA1538 -                                                                              TA1538 +                                 STRAIN  S9         S9        S9      S9                                       ______________________________________                                        Dose                                                                          μg/plate                                                                   0       126         123      15      19                                               n = 41      n = 56    n = 30 n = 42                                   5       292        -24       10      21                                               n = 3      n = 3     n = 3   n = 3                                    10      337        -22       12      22                                               n = 3      n = 3     n = 3   n = 3                                    Positive                                                                              1.5 μg/plate              5 μg/plate                            Control Sodium                       2-Amino-                                         Azide                        fluorene                                         965                          1064                                             n = 38                       n = 30                                   ______________________________________                                    

                  TABLE 15(A)                                                     ______________________________________                                        Compound 2                                                                    STRAIN                                                                        Dose      TA98      TA98      TA100  TA100                                    μg/plate                                                                             -S9       +S9       -S9    +S9                                      ______________________________________                                         0        20        25        126    123                                                n = 35    n = 50    n = 41 n = 56                                    5                            103    -18                                                                    n = 3  n = 3                                     10       28        24        46     1                                                  n = 3     n = 3     n = 6  n = 6                                     50       52        35        182    115                                                n = 3     n = 3     n = 3  n = 3                                    100       39        53        121    96                                                 n = 6     n = 6     n = 3  n = 3                                    250       29        69                                                                  n = 3     n = 3                                                     500       6         63                                                                  n = 3     n = 3                                                     Positive  10 μg/plt                                                                            10 μg/plt     5 μg/plate                            Control   9-Amino-  2-Amino-         2-Amino-                                           acridine  fluorene         fluorene                                           284       73               1064                                               n = 6     n = 24           n = 30                                   ______________________________________                                    

                  TABLE 15(B)                                                     ______________________________________                                        Compound 2                                                                    STRAIN                                                                        Dose     TA1537    TA1537    TA1538  TA1538                                   μg/plate                                                                            -S9       +S9       -S9     +S9                                      ______________________________________                                         0       9         9         15      19                                                n = 30    n = 45    n = 30  n = 42                                   5                            -8      2                                                                     n = 3   n = 3                                    10       36        5         -13     4                                                 n = 3     n = 3     n = 3   n = 3                                    50       282       40                                                                  n = 3     n = 3                                                      100      258       88                                                                  n = 3     n = 3                                                      250      176       744                                                                 n = 3     n = 3                                                      500      114       395                                                                 n = 3     n = 3                                                      Positive 10 μg/plt                                                                            10 μg/plt      5 μg/plate                            Control  9-Amino-  2-Amino-          2-Amino-                                          acridine  fluorene          fluorene                                          284       73                1064                                              n = 6     n = 24            n = 30                                   ______________________________________                                    

                  TABLE 16                                                        ______________________________________                                        Compound 3                                                                    STRAIN                                                                        Dose      TA100     TA100    TA1538  TA1538                                   μg/plate                                                                             -S9       +S9      -S9     +S9                                      ______________________________________                                         0        126       123      15      19                                                 n = 41    n = 56   n = 30  n = 42                                    5        47        -19      0       1                                                  n = 3     n = 3    n = 3   n = 3                                    10        47        8        -6      9                                                  n = 3     n = 3    n = 3   n = 3                                    Positive  1.5 μg/plt              5 μg/plt                              Control   Sodium                     2-Amino-                                           Azide                      fluorene                                           965                        1064                                               n = 38                     n = 30                                   ______________________________________                                    

                  TABLE 17                                                        ______________________________________                                        Compound 4                                                                    STRAIN                                                                        Dose   TA100      TA100     TA1538  TA1538                                    μg/plate                                                                          -S9        +S9       -S9     +S9                                       ______________________________________                                        0      126        123       15      19                                               n = 41     n = 56    n = 30  n = 42                                    5      -41        -10       -2      7                                                n = 3      n = 3     n = 3   n = 3                                     10      3         -3        -2      -2                                               n = 3      n = 3     n = 3   n = 3                                     Positive                                                                             1.5 μg/plate              5 μg/plate                             Control                                                                              Sodium                       Amino-                                           Azide                        fluorene                                         965                          1064                                             n = 38                       n = 30                                    ______________________________________                                    

                  TABLE                                                           ______________________________________                                        STRAIN                                                                        Dose    TA98     TA98       TA100     TA100                                   μg/plate                                                                           -S9      +S9        -S9       +S9                                     ______________________________________                                         0      20       25         126       123                                             n = 38   n = 53     n = 41    n = 56                                   5                          -32       12                                                                  n = 3     n = 3                                    10     12       -5         3         -5                                              n = 3    n = 3      n = 9     n = 9                                    50     12       2          2         24                                              n = 3    n = 3      n = 6     n = 6                                   100     22       20         -18       -2                                              n = 6    n = 6      n = 6     n = 6                                   250     12       40                   -38                                             n = 3    n = 3                n = 3                                   500     9        2                                                                    n = 3    n = 3                                                        Positive         5 μg/plate                                                                            1.5 μg/plate                                   Control          2-Amino-   Sodium                                                             fluorene   Azide                                                              1154       965                                                                n = 35     n = 38                                            ______________________________________                                    

                  TABLE 18(B)                                                     ______________________________________                                        Compound 6                                                                    STRAIN                                                                        Dose    TA1537    TA1537     TA1538 TA1538                                    μg/plate                                                                           -S9       +S9        -S9    +S9                                       ______________________________________                                         0      9         9          15     19                                                n = 30    n = 45     n = 30 n = 42                                     5                           -5     0                                                                      n = 3  n = 3                                      10     141       -1         -2     8                                                 n = 6     n = 6      n = 3  n = 3                                      50     2010      17                                                                  n = 6     n = 6                                                       100     795       35                                                                  n = 6     n = 6                                                       250     228       99                                                                  n = 6     n = 6                                                       500     43        369                                                                 n = 3     n = 3                                                       Positive                                                                              10 μg/plate                                                                          10 μg/plate    5 μg/plate                             Control 9-Amino-  2-Amino-          2-Amino-                                          acridine  fluorene          fluorene                                          284       73                1064                                              n = 6     n = 24            n = 30                                    ______________________________________                                    

                  TABLE 19(A)                                                     ______________________________________                                        Compound 18                                                                   STRAIN                                                                        Dose           TA98        TA98                                               μg/plate    -S9         +S9                                                ______________________________________                                         0             17          28                                                                n = 3       n = 3                                               5                                                                             10            21          8                                                                 n = 3       n = 3                                               50            303         6                                                                 n = 3       n = 3                                              100            390         26                                                                n = 6       n = 6                                              200            225         42                                                                n = 3       n = 3                                              500                                                                           Positive                   5 μg/plate                                      Control                    2-Amino-                                                                      fluorene                                                                      2589                                                                          n = 3                                              ______________________________________                                    

                  TABLE 19(B)                                                     ______________________________________                                        Compound 18                                                                   STRAIN                                                                        Dose         TA1537        TA1537                                             μg/plate  -S9           +S9                                                ______________________________________                                         0           8             7                                                               n = 3         n = 3                                               5                                                                             10          21            8                                                               n = 3         n = 3                                               50          303           6                                                               n = 3         n = 3                                              100          390           26                                                              n = 3         n = 3                                              200          225           42                                                              n = 3         n = 3                                              500                                                                                        100 μg/plate                                                                             100 μg/plate                                                 AMT           AMT                                                             608           500                                                             n = 3         n = 3                                              ______________________________________                                    

Maron and Ames (1983) describe the conflicting views with regard to thestatistical treatment of data generated from the test. In light of this,this example adopts the simple model of mutagenicity being characterizedby a two-fold or greater increase in the number of revertants abovebackground (in bold in the tables), as well as dose dependent mutagenicresponse to drug.

With regard to 8-MOP, the only mutagenic response detected was a weakbase-substitution mutagen in TA102 at 500 μg/plate (TABLE 13(B)).

In sharp contrast, AMT (TABLE 12(A) and 12(B)) showed frameshiftmutagenicity at between 5 and 10 μg/plate in TA97a and TA98, at 5μg/plate in TA1537 and at 1 μg/plate in TA1538. AMT showed nosignificant base-substitution mutations.

Looking at Compound 1, the only mutagenic response detected was a weakframeshift mutagen in TA1538 at 5 μg/plate in the presence of S9.Compound 1 also displayed mutation in the TA100 strain, but only in theabsence of S9. Compound 2 also showed weak frameshift mutagenicity inthe presence of S9 in TA98 and TA1537. Compounds 3 and 4 showed nomutagenicity. Compound 6 had no base substitution mutagenicity, butshowed a frameshift response in TA98 in the presence of S9 atconcentrations of 250 μg/plate and above. It also showed a response at50 μg/plate in TA1537 in the presence of S9. Compound 18 showed only aweak response at high concentrations in the presence of S9 in strains TA90 and TA 1537. The response was higher in the absence of S9, but stillwas significantly below that of AMT, which displayed mutagenicity atmuch lower concentrations (5 μg/plate).

From this data it is clear that the compounds of the present inventionare less mutagenic than AMT, as defined by the Ames test. At the sametime, these compounds show much higher inactivation efficiency than8-MOP, as shown in Examples 12 and 16. These two factors support thatthe compounds of the present invention combine the best features of bothAMT and 8-MOP, high inactivation efficiency and low mutagenicity.

EXAMPLE 18

In Example 15, the compounds of the present invention exhibited theability to inactivate pathogens in synthetic media. This exampledescribes methods by which synthetic media and compounds of the presentinvention may be introduced and used for inactivating pathogens inblood. FIG. 25A schematically shows the standard blood productseparation approach used presently in blood banks. Three bags areintegrated by flexible tubing to create a blood transfer set (200)(e.g., commercially available from Baxter, Deerfield, Ill.). After bloodis drawn into the first bag (201), the entire set is processed bycentrifugation (e.g., Sorvall™ swing bucket centrifuge, Dupont),resulting in packed red cells and platelet rich plasma in the first bag(201). The plasma is expressed off of the first bag (201) (e.g., using aFenwall™ device for plasma expression), through the tubing and into thesecond bag (202). The first bag (201) is then detached and the two bagset is centrifuged to create platelet concentrate and platelet-poorplasma; the latter is expressed off of the second bag (202) into thethird bag (203).

FIG. 25B schematically shows an embodiment of the present invention bywhich synthetic media and photoactivation compound are introduced toplatelet concentrate prepared as in FIG. 25A. A two bag set (300) issterile docked with the platelet concentrate bag (202) (indicated as"P.C."). Sterile docking is well-known to the art. See e.g., U.S. Pat.No. 4,412,835 to D. W. C. Spencer, hereby incorporated by reference. Seealso U.S. Pat. Nos. 4,157,723 and 4,265,280, hereby incorporated byreference. Sterile docking devices are commercially available (e.g.,Terumo, Japan).

One of the bags (301) of the two bag set (300) contains a syntheticmedia formulation of the present invention (indicated as "STERILYTE").In the second step shown in FIG. 25B, the platelet concentrate is mixedwith the synthetic media by transferring the platelet concentrate to thesynthetic media bag (301) by expressing the platelet concentrate fromthe first blood bag into the second blood bag via a sterile connectionmeans. The photoactivation compound can be in the bag containingsynthetic media (301), added at the point of manufacture. Alternatively,the compound can be mixed with the blood at the point of collection, ifthe compound is added to the blood collection bag (FIG. 25A, 201) at thepoint of manufacture. The compound may be either in dry form or in asolution compatable with the maintainance of blood.

FIG. 25C schematically shows one embodiment of the decontaminationapproach of the present invention applied specifically to plateletconcentrate diluted with synthetic media as in FIG. 25B. In thisembodiment, platelets have been transferred to a synthetic media bag(301). The photoactivation compound either has already been introducedin the blood collection bag (201) or is present in the synthetic mediabag (301). Either the platelets are then expressed into the syntheticmedia bag via a sterile connection means (as shown) or the syntheticmedia is expressed into the platelet bag. The bag containing the mixtureof platelet concentrate and synthetic media (301), which has UV lighttransmission properties and other characteristics suited for the presentinvention, is then placed in a device (such as that described in Example1, above) and illuminated.

Following phototreatment, the decontaminated platelets are transferredfrom the synthetic media bag (301) into the storage bag (302) of the twobag set (300). The storage bag can be a commercially available storagebag (e.g., CLX bag from Cutter).

FIG. 25D schematically shows an embodiment of the decontaminationapproach of the present invention, which includes a capture device toremove photoinactivation compound from the treated material afterphototreatment. The present invention contemplates several adsorptivematerials which may be used in a capture device to removephotoinactivation compounds, of which the following is a nonexclusivelist: Amberlite XAD-4 (available from Rohm and Haas Ltd., Croydon,Surrey, UK) ("Resin hemoperfusion for Acute Drug Intoxication," ArchIntern Med 136:263 (1976)); Amberlite XAD-7 ("Albumin-Coated AmberliteXAD-7 Resin for Hemoperfusion in Acute Liver Failure," ArtificialOrgans, 3:20 (1979); Amberlite 200, Amberlite DP-1, Amberlite XAD-2,Amberlite XAD-16; activated charcoals, ("Charcoal haemoperfusion in DrugIntoxication," British J. Hospital Med. 49:493 (1993); silica ("In vitroStudies of the Efficacy of Reversed Phase Silica Gel as a Sorbent forHemo- and Plasmaperfusion," Clinical Toxicology 30:69 (1992)). In oneembodiment, the present invention contemplates an absorptive materialoperating in conjunction with a filtering means to remove compounds.

EXAMPLE 19

This example involves an assessment of the impact of the compounds andmethods of the present invention on platelet function. Four indicatorsof platelet viability and function were employed: 1) GMP-140 expression;2) maintenance of pH; 3) platelet aggregation, and 4) platelet count.

To measure the effects of the present compounds and methods ofdecontamination on platelet function using these four indicators, foursamples were prepared for each compound tested, two control samples andtwo containing compound. Three units of human platelets were obtainedfrom the Sacramento Blood Center, Sacramento, Calif. These were eachtransferred under sterile conditions to 50 ml centrifuge tubes, thenaliquots of each unit were transferred into a second set of 50 mlsterile centrifuge tubes. To each centrifuge tube containing plateletconcentrate (PC), an aliquot of compound stock was added to reach afinal concentration of 100 μM of compound. The compounds tested in thisexperiment were Compound 2 (36 μL of 10 mM stock added to 4 ml PC),Compound 6 (173.5 μl of 9.8 mM stock added to 16.8 ml PC), Compound 17(2.0 ml of 1 mM stock added to 18 ml PC) and Compound 18 (0.842 ml of2.0 mM stock to 16 ml PC). The samples were pipetted gently up and downto mix. Then aliquots (either 3 ml or 8 ml) of each sample wastransferred to two sterile Teflon™ Medi-bags™ (American Fluoroseal Co.,Silver Springs, Md.) (presently owned by The West Company, Lionville,Pa.). Samples were treated in one of two different sized bags, havingeither 3 ml or 8 ml capacity. The bags both have approximately the samesurface area to volume ratios, and previous experiments have shown thatthe two bags exhibit approximately equivalent properties duringirradiation of samples. (Data not shown). For each compound tested, twocontrol samples without compound were prepared by again removingaliquots of platelet concentrate (17 ml if using an 8 ml bag, 4 ml ifusing a 3 ml bag) from the same one of the first set of 50 ml centrifugetubes from which the compound sample was drawn, and dividing intoMedibags, as before. One of each pair of Medibags containing a compound,and one of each pair of control Medibags, were illuminated for 5Joules/cm² on the device described in Example 1, above. Then allexperimental and control Medibags were placed on a platelet shaker forstorage for 5 days. The same experiments were repeated several times toobtain more statistically meaningful data, as represented by "n", thenumber of data points represented, in the graphs of FIGS. 26-29,discussed below. Also in FIGS. 26-29, "C1" represents an untreatedcontrol at day 1, "D5" represents an untreated control after a five daystorage, "uv" represents a sample which was treated with ultravioletlight only, and "PCD" represents the test sample, treated withultraviolet light and a compound of the present invention.

To obtain data for control samples at day one, approximately 3 ml wereremoved from the remaining volume of each of the three units and dividedinto two 1.5 ml tubes. These samples were tested for pH as describedbelow. A platelet count was also taken, as described below, at a 1:3dilution. The residual platelet concentrate from each unit was spun for10 minutes at 3800 rpm (3000 g) in Sorval RC3B (DuPont Company,Wilmington, Del.) to pellet platelets. Plasma was then decanted into 2sterile 50 ml tubes (one for Day one, and the other stored at 4° C. forDay 5) for use in the aggregation assay.

1) GMP-140 Expression

When platelets become activated, an alpha granule membrane glycoproteincalled p-selectin (GMP140) becomes exposed on the platelet surface. Lessthan (5%) of fresh, normal unstimulated platelets express detectableGMP140 levels by flow cytometry. See generally M. J. Metzelaar, Studieson the Expression of Activation-Markers on Human Platelets (Thesis1991).

To measure GMP140, a small aliquot of platelet rich plasma is placed inHEPES buffer containing a GMP140-binding antibody or an isotype controlmouse IgG. CD62 is a commercially available monoclonal antibody whichbinds to GMP140 (available from Sanbio, Uden, the Netherlands; CaltagLabs, So. San Francisco, Calif., and Becton Dickinson, Mountain View,Calif.). After a fifteen minute incubation at room temperature, GoatF(ab')2 Anti-Mouse IgG conjugated to FITC (Caltag Laboratories, So. SanFrancisco, Calif.) is added to the tube in saturating amounts andallowed to incubate at room temperature (RT) for 15 minutes. Finally,the cells are diluted in 1% paraformaldehyde in phosphate bufferedsaline and analyzed on a FACSCAN™ (Becton Dickinson, Mountain View,Calif.). The positive control is made by adding Phorbol MyristateAcetate (PMA) to the test system at a final concentration of 2×10⁻⁷ M.

In this example, CD62 was employed to measure the impact, if any, ofirradiation in the presence of several compounds of the presentinvention on platelet activation. The antibody was mixed with HEPESbuffer (10 μL antibody [0.1 mg/ml]: 2.49 mL buffer) and stored in 50 μLaliquots at -40° C. prior to use. A positive control consisted of: 10 μLCD62, 8 μL PMA and 2.482 mL Hepes buffer. A mouse IgG1 control (0.05mg/ml) (Becton Dickinson, Mountain View, Calif. #9040) 5× concentratedwas also employed. The antibody was diluted in HEPES buffer (20 μLantibody: 2.48 ml buffer) and stored at -40° C. Phorbol MyristateAcetate (PMA) (Sigma, St. Louis, Mo.) was stored at -40° C. At time ofuse, this was dissolved in DMSO (working concentration was 10 μg/mL).

1% Paraformaldehyde (PFA) (Sigma, St. Louis, Mo.) was prepared by adding10 grams paraformaldehyde to 1 liter PBS. This was heated to 70° C.,whereupon 3M NaOH was added dropwise until the solution was clear. Thesolution was cooled and the pH was adjusted to 7.4 with 1N HCl. This wasfiltered and stored.

Processing each of the samples of platelet concentrate after treatmentinvolved adding 5 microliters of platelet concentrate, diluted 1:3 inHepes buffer, to each microcentrifuge tube containing the antibody CD62,and appropriate reagents and mixing very gently by vortex. The sampleswere then incubated for 15 minutes at room temperature.

The Goat anti-Mouse IgG-FITC (diluted 1:10 in HEPES buffer) was added (5microliters) to each tube and the solution was mixed by gentle vortex.The samples were incubated for an additional 15 minutes at roomtemperature. Next, 1 ml of 1% PFA in PBS was added to each tube andmixed gently. The platelets were analyzed on the FACSCAN™. The resultsare shown in FIGS. 26C, 27C, 28C, and 29C. (FIGS. 26 correspond toCompound 2, FIGS. 27 correspond to Compound 6, FIGS. 28 correspond toCompound 17 and FIGS. 29 correspond to Compound 18). Clearly, three ofthe four compounds tested, 2, 6, and 17, exhibited little or nodifference between the day 5 untreated control (D5) and the sampletreated with both light and psoralen compound (PCD). Only Compound 18exhibited a notable increase above the control. But the value was stillwell below the positive control value.

2) Maintainance of pH:

Changes in pH of platelets in concentrate can alter their morphologicalcharacteristics and their survival post transfusion. Moroff, G., et al.,"Factors Influencing Changes in pH during Storage of PlateletConcentrates at 20°-24° C.," Vox Sang. 42:33 (1982). The range of pH atwhich platelets function normally is from approximately 6.0-6.5 to 7.6.Stack, G. and E. L. Snyder, "Storage of Platelet Concentrate," BloodSeparation and Platelet Fractionation 99, at 107 (1991). To measure pHof the samples, a CIBA-CORNING 238 pH/Blood Gas analyzer was used(CIBA-CORNING, Norwood, Me.). A small amount of platelet concentratefrom each sample was introduced into the pH/Blood Gas analyzer.

Measurements of pH were taken at time zero and after 5 days of storagefor all samples. FIGS. 26D, 27D, 28D and 29D are bar graphs showing pHresults for a dark control, a light control and an experimental lightplus compound. These graphs indicate that the pH of platelet concentratesamples after illumination in the presence of any one of the compoundsremains above a pH of 6.5. Thus platelets remain at a pH acceptable forstored platelets following photoinactivating treatment using compoundsof the present invention.

3) Aggregation

Platelet aggregation is measured by the change in optical transmissionthat a platelet sample exhibits upon stimulation of aggregation.Platelet aggregation was measured using a Whole Blood Aggregometer(Chrono-Log Corp., Havertown, Pa., model 560VS). The number of plateletsin each sample was controlled to be constant for every measurement. AModel F800 Sysmex cell counter (Toa Medical Electronics, Kobe, Japan)was used to measure platelet count in the platelet samples andautologous plasma was used to adjust platelet counts to 300,000/mL ofplatelet concentrate.

For the procedure, all the samples were incubated in a capped plastictube for 30 minutes at 37° C. for activation. The aggregometer waswarmed up to 37° C. The optical channel was used for plateletaggregation measurement. The magnetic speed of the aggregometer was setat 600/min. Remaining platelet concentrate, from the units obtainedwhich was not drawn as a sample for treatment, was centrifuged at highspeed (14,000 g) with a micro-centrifuge for 5 minutes to obtaincontainers of platelet poor plasma autologous to the experimentalsamples.

To begin, 0.45 ml of the autologous platelet poor plasma was added alongwith 0.5 ml of saline into a glass cuvette and placed in the PPPchannel. Then 0.45 ml of the sample platelet concentrate and 0.50 ml ofsaline were added to a glass cuvette (containing a small magnet) intothe sample channel. After one minute, ADP and collagen reagents (10 μl)each were added to the sample cuvette. The final concentration of ADPwas 10 μM and the final concentration of collagen was 5 μg/ml. Plateletaggregation was recorded for about 8-10 minutes or until the maximumreading was reached.

The results appear in FIGS. 26B, 27B, 28B, and 29B. The 100% aggregationline is the level at which the recorder was set to zero. The 0%aggregation line is where the platelets transmitted before the ADP andcollagen were added. The aggregation value for the sample is determinedby taking the maximum aggregation value as a percent of the total range.Three of the four compounds tested showed very little or no differencein aggregation levels between the samples treated with compound and theuntreated samples which were stored for 5 days. Compound 2 exhibited asmall reduction in aggregation, of approximately 8% from the day 1control. The aggregation for the sample treated with compound and uv wasthe same as that for the uv only sample. This is supporting evidencethat the decontamination compounds tested do not have a significanteffect on platelet aggregation when used in the methods of the presentinvention.

4) Count

A Sysmex cell counter was used to measure platelet count in the plateletsamples. Samples were diluted 1:3 in blood bank saline.

The results of the platelet count measurements appear in FIGS. 26A, 27A,28A, and 29A. For each of the compounds, the samples show little or nodrop in platelet count between the Day 5 control and the Day 5 treatedsample. Interestingly, samples treated with Compounds 6, 17 and 18 alldisplay a higher platelet count than samples treated with light alone.For example, samples treated with Compound 6 had counts equivalent tothe 5 day control, but samples treated with only ultraviolet lightshowed approximately a 33% reduction in platelet count. Thus, not onlyis treatment with compounds of the present invention compatible with themaintenance of platelet count, but it actually appears to prevent thedrop in count caused by ultraviolet light exposure.

EXAMPLE 20

A preferred compound for decontaminating blood subsequently used in vivoshould not be mutagenic to the recipient of the blood. In the first partof this experiment, some compounds were screened to determine theirgenotoxicity level in comparison to aminomethyltrimethylpsoralen. In thesecond part, the in vivo clastogenicity of some compounds of the presentinvention was measured by looking for micronucleus formation in mousereticulocytes.

1) Genotoxicity

Mammalian cell cultures are valuable tools for assessing the clastogenicpotential of chemicals. In such studies, cells are exposed to chemicalswith and/or without rat S-9 metabolic activation system (S-9) and arelater examined for either cell survival (for a genotoxicity screen) orfor changes in chromosome structure (for a chromosome aberration assay).

Chinese hamster ovary (CHO; ATCC CCL 61 CHO-K1, proline-requiring) cellswere used for the in vitro genotoxicity and chromosomal aberrationtests. CHO cells are used extensively for cytogenic testing because theyhave a relatively low number of chromosomes (2n=20) and a rapid rate ofmultiplication (˜12 to 14 hours, depending on culture conditions). Thecells were grown in an atmosphere of 5% CO₂ at approximately 37° C. inMcCoy's 5a medium with 15% fetal bovine serum (FBS), 2 mM L-glutamine,and 1% penicillin-streptomycin solution to maintain exponential growth.This medium was also used during exposure of the cells to the testcompound when no S-9 was used. Cell cultures were maintained and cellexposures were performed in T-75 or T-25 flasks.

Each of the sample compounds were tested at seven dilutions, 1, 3, 10,33, 100, 333, and 1000 μg/ml. The compound was added in complete McCoy's5a medium. After the compound was added, cells were grown in the dark atapproximately 37° C. for approximately 3 hours. The medium containingthe test compound was then aspirated, the cells were washed three timeswith phosphate-buffered saline (PBS) at approximately 37° C., and freshcomplete McCoy's 5a medium was added. The positive control wasmethylmethane sulfonate. The solvent control was dimethylsulfoxide(DMSO) diluted in culture medium. For assays using metabolic activation(see below) the activation mixture was also added to the solventcontrol. The cultures were then incubated for an additional time ofapproximately 12 hours before they were harvested. Colchicine (finalconcentration, 0.4 μg/ml) was added approximately 2.5 hours prior to theharvest.

After approximately 2.5 hours in colchicine, the cells were harvested.Cells were removed from the surface of the flasks using a cell scraper.The resulting cell suspension was centrifuged, the supernatant,aspirated, and 4 ml of a hypotonic solution of 0.075M KCI added to thecells for 15 minutes at approximately 37° C. The cells were thencentrifuged, the supernatant aspirated, and the cells suspended in afixative of methanol: acetic acid (3:1). After three changes offixative, air-dried slides were prepared using cells from all flasks.The cell density and metaphase quality on the initial slide from eachflask was monitored using a phase-contrast microscope; at least twoslides of appropriate cell density were prepared from each flask. Theslides were stained in 3% Giemsa for 20 min, rinsed in deionized water,and passed through xylene. Coverslips were mounted with Permount. Slidesare then examined to determine what concentration of each test compoundrepresented a toxic dose.

An analysis of the results showed that AMT was genotoxic at 30 μg/ml. Incontrast, Compounds 2 and 6 were only genotoxic at 100 μg/ml, more thanthree times the toxic dose of AMT.

A psoralen compound with a structure distinct from compounds of thepresent invention, 8-aminomethyl-4,4',5'-trimethylpsoralen, was alsotested in this experiment and proved to be toxic at 10 μg/ml. While the8-substituted aminomethyl compound and similar structures may not besuited for methods of the present invention, they may be useful foralternative purposes. In light of the ability of the compounds toprevent nucleic acid replication, in combination with their extremetoxicity, the compounds could be used, for example, to treat diseasescharacterized by uncontrolled cell growth, such as cancer.

2) Micronucleus Assay Protocol

Saline solutions were prepared for Compounds 2, 6, 17 and 18 at variousconcentrations. Male Balb/c mice were then injected with 0.1 ml of acompound solution via the tail vein. At least 3 mice were injected perdose level. Saline only was used as a negative control. For a positivecontrol, cyclophosphamide (cycloPP) was administered at a dose of 30mg/kg. In the experimental group, the injections were repeated once perday for four days. In the positive control group, the sample wasadministered only once, on day three. On day 5, several microliters ofblood were withdrawn from each subject and smeared on a glass slide.Cells were fixed in absolute methanol and stored in a slide rack.

For analysis, cells were stained with acridine orange and visualizedunder a fluorescence microscope by counting: (i) the number ofreticulocytes per 5000 erythrocytes; and (ii) the number ofmicronucleated reticulocytes per 1000 reticulocytes. Reticulocytes weredistinguished by their red fluorescence due to the presence of RNA.Micronuclei were distinguished by their green fluorescence due to thepresence of DNA. The percentage of reticulocytes (% PCE) was thencalculated. A decrease in the frequency of erythrocytes, represented byan increase in the percentage of reticulocytes, is an indication of bonemarrow toxicity. The percentage of reticulocytes with micronuclei (% PCEwith MN) was also calculated. An increase in % PCE with MN is a measureof clastogenicity.

After initial results were determined, the experiment was repeated usingincreased dose levels, until: (i) Micronucleus formation was seen; or(ii) Bone marrow toxicity was observed; or (ii) The lethal dose wasreached; or (iv) A dose of 5 g/kg was administered. For the assays witheach of the compounds 2, 6, 17 and 18, the acutely lethal dose wasreached before there were any signs of bone marrow toxicity ormicronucleus formation. The results of the experiment appear in Table20, below. As is clear from the table, no bone marrow toxicity wasobserved for any of the compounds at the doses tested. The percentreticulocyte value for treatment with each compound remained close tothe negative control value. This is in contrast with a drop ofapproximately 2-2.5% PCE/RBC seen in the positive control, representingerythrocyte depletion due to bone marrow toxicity. Nor did any of thecompounds display clastogenic action.

                  TABLE 20                                                        ______________________________________                                                  DOSE     PCE/RBC   PCE + MN #                                       COMPOUND  (mg/kg)  (%)       (%)      duplicate                               ______________________________________                                        2         40       3.08 ± 0.82                                                                          0.20 ± 0.14                                                                         4                                       2         25       3.46 ± 0.32                                                                          0.25 ± 0.11                                                                         6                                       CycloPP   30       1.65 ± 0.64                                                                          1.98 ± 0.40                                                                         6                                       saline             3.49 ± 0.55                                                                          0.18 ± 0.13                                                                         6                                       6         45       3.79 ± 0.41                                                                          0.36 ± 0.14                                                                         3                                       6         30       3.61 ± 0.12                                                                          0.27 ± 0.38                                                                         3                                       17        45        5.7 ± 2.14                                                                          0.31 ± 0.07                                                                         3                                       17        30       3.47 ± 0.83                                                                          0.30 ± 0.17                                                                         3                                       CycloPP   30       0.99 ± 0.33                                                                          1.76 ± 0.64                                                                         3                                       saline             3.47 ± 0.44                                                                          0.23 ± 0.15                                                                         3                                       18        20       3.48 ± 0.79                                                                          0.17 ± 0.06                                                                         3                                       18        7.5      3.59 ± 0.33                                                                          0.43 ± 0.12                                                                         3                                       18        3.75     3.61 ± 1.14                                                                          0.17 ± 0.12                                                                         3                                       CycloPP   30       1.39 ± 0.41                                                                          2.09 ± 0.17                                                                         3                                       saline             3.31 ± 0.63                                                                          0.36 ± 0.11                                                                         3                                       ______________________________________                                    

EXAMPLE 21

In EXAMPLE 13, the inactivation of cell-free HIV virus, using compoundsand methods of the present invention, is shown. This example showsinactivation of cell-associated HIV also using compounds of the presentinvention.

H9 cells chronically infected with HIV_(IIIB) were used. (H9/HTLV-III-BNIH 1983 Cat.#400). Cultures of these cells were maintained in highglucose Dulbecco Modified Eagle Medium supplemented with 2 mML-glutamine, 200 u/mL penicillin, 200 μg/ml streptomycin, and 9% fetalbovine serum (Intergen Company, Purchase, N.Y.) For maintenence, theculture was split once a week, to a density of 3×10⁵ to 4×10⁵ cells/mland about four days after splitting, 3.3% sodium bicarbonate was addedas needed. For the inactivation procedure, the cells were used threedays after they were split. They were pelleted from their culture mediumat 400 g×10 minutes, the supernatant was discarded, and the cells wereresuspended in one to five day old human platelet concentrate (PC) (pH7.5-6.5), to a concentration of 2×10⁶ cells/ml. Aliquots of thePC-infected cell suspension were made for psoralen free dark controls,for sporalen free UVA only controls, for psoralen dark controls, and forthe psoralen plus UVA experimental sample. Concentratedfilter-sterilized stock solutions of each psoralen in water were dilutedinto the appropriate aliquots to yield a final concentration of 150 μM.(A 10 mM stock of Compound 18 was diluted about 67-fold and a 2 mM stockof Compound 2 was diluted about 13-fold). After an equilibration periodof thirty minutes at room temperature, 0.5 ml of each of the darkcontrols was placed in a cryovial and stored in the dark at -80° C. ForUVA illumination, 8 ml of the psoralen free aliquot and 8 ml of eachpsoralen containing aliquot were introduced into a modified F1 20Teflon™ bag (modified to be 92 cm² total surface area, The West Co.,Phoenixvill, Pa.) via a plastic disposable 10 ml syringe attached to oneof the polypropylne ports on the bag. This gave an average path lengthof 0.17 cm. The bags were then illuminated for a total exposure of 3Joules/cm² in the device described in Example 1, above, attached to acirculating refrigerating waterbath set at 4° C., which maintains thetemperature in the bag at approximately 22°-25° C. During exposure, thedevice was shaken on a platelet shaker (Helmer Labs, Noblesville, Ind.).After exposure, the contents of the bags were withdrawn by a freshsyringe through the remaining unused port on the bag, and placed incryovials for storage in the dark at -80° C. until analysis.

The stored samples were thawed at 37° C., then titrated in an HIVmicroplaque assay, as described in Hanson, C. V., Crawford-Miksza, L.and Sheppard, H. W., J. Clin. Micro 28:2030 (1990), and as described inEXAMPLE 13, above, with the following modifications. Clot removal fromeach sample was performed before plating. Because plating of a targetvolume of 4 ml after clot removal was desired, an excess of sample (6ml) was transferred to a polypropylene tube and diluted to a finalvolume of 60 ml with Test and control samples from the inactivationprocedure were diluted in 50% assay medium and 50% normal human pooledplasma. The samples were serially diluted directly in 96-well plates(Corning Glass Works, Corning, N.Y.). The plates were mixed on anoscillatory shaker for 30 seconds and incubated at 37° C. in a 5% CO₂atmosphere for 1 to 18 hours. MT-2 cells (0.025 mL) [clone alpha-4,available (catalog number 237) from the National Institutes of HealthAIDS Research and Reference Reagent Program, Rockville, Md.] were addedto each well to give a concentration of 80,000 cells per well. After anadditional 1 hour of incubation at 37° C. in 5% CO₂, 0.075 mL of assaymedium containing 1.6% SeaPlaque agarose (FMC Bioproducts, Rockland,Me.) and prewarmed to 38.5° C. was added to each well. The plates werekept at 37° C. for a few minutes until several plates had accumulatedand then centrifuged in plate carriers at 600×g for 20 minutes in acentrifuge precooled to 10° C. In the centrifuge, cell monolayers formedprior to gelling of the agarose layer. The plates were incubated for 5days at 37° C. in 5% CO₂ and stained by the addition of 0.05 mL of 50μg/mL propidium iodide (Sigma Chemical Co.) in phosphate-buffered saline(pH 7.4) to each well. After 24 to 48 hours, the redfluorescence-stained microplaques were visualized by placing the plateson an 8,000 μW/cm² 304 nm UV light box (Fotodyne, Inc., New Berlin,Wis.). The plaques were counted at a magnification of ×20 to ×25 througha stereomicroscope.

The results were as follows: Compound 2 (150 μM) inactivated >6.7 logsof HIV after 3 Joules/cm² irradiation (compared to dark and lightcontrols of 0 log inactivation, starting log titer 6.1 plaque formingunits/ml). At the same concentration and irradiation time, Compound 18inactivated >7.2 logs of HIV (compared to a dark control of 0 logs and alight control of 0.1 logs, starting titer 6.6). This example supportsthat the compounds of the present invention are effective ininactivating cell associated virus.

EXAMPLE 22

This example involves an assessment of new synthetic media formulationsas measured by the following in vitro platelet function assays: 1)maintenance of pH; 2) platelet aggregation ("Agg") and 3) GMP140expression. The assays for each of these tests have been describedabove.

                  TABLE 21*                                                       ______________________________________                                                  S 2.19                                                                              S 2.22      S 3.0  S 4.0                                      ______________________________________                                        Na gluconate                                                                              23       0           0    0                                       Na acetate  27      20          20   20                                       glucose      0       2           2    2                                       mannitol    30      20           0   20                                       KCl          5       4           4    4                                       NaCl        45      80          100  90                                       Na.sub.3 citrate                                                                          15      15          10   10                                       Na phosphate                                                                              20      20          20   20                                       MgCl.sub.2   0       3           2    2                                       ______________________________________                                         *Amounts in mM                                                           

Four formulations were prepared: S 2.19, S 2.22, S 3.0 and S.4.0. Thecomposition of these synthetic media formulations are shown in Table 21.One unit of human platelet rich plasma (PRP) was obtained from theSacramento Blood Bank. The unit was centrifuged at room temperature for6 minutes at 4000 rpm and then transferred to a unit press. Using anattached transfer line, plasma was expressed from the unit, leavingapproximately 9.4 mls of residual plasma.

The unit was allowed to rest for 1 hour, after which it was gentlykneaded to resuspend the platelets. To 0.6 ml of the suspension, 2.4 mlof plasma was added back and the entire contents transferred to aTeflon™ minibag. The reconstituted unit was assayed for pH and othertests the next day, with the following results:

    ______________________________________                                                pH     7.19                                                                   GMP140 62%                                                                    Agg    58%                                                            ______________________________________                                    

The remaining unit was then used to evaluate synthetic media forplatelet storage with and without photodecontamination. Aliquots (0.8ml) from the unit were added to each formulation (3.2 mls) in tubes. 3mls of each mixture was transferred to a Teflon™ minibag (final plasmaconcentration of 20%).

Five days later, platelet function was assessed using the battery oftests described above. The results for each of the synthetic mediaformulations are shown in Table 22 below.

                  TABLE 22                                                        ______________________________________                                                no light         light                                                        S 2.19                                                                              S 2.22     S 2.19  S 2.22                                       ______________________________________                                        pH        6.86    6.82       6.83  6.60                                       GMP140    87%     74%        90%   80%                                        Agg       30      48         16    31                                         ______________________________________                                    

It appeared that the synthetic media containing 2 mM glucose (i.e., S2.22) maintained platelet function, as measured by GMP140 andAggregation, better than the synthetic media that did not containglucose (i.e., S 2.19).

To confirm the above finding, experiments were repeated ("n" being thenumber of replicate experiments) with these formulations as well asadditional glucose-free formations (3.0 and 4.0). Platelet function wasevaluated both before and after storage, and in conjunction withphotodecontamination. A summary of the results is provided in Tables 4,5 and 6 below.

                  TABLE 23*                                                       ______________________________________                                        Plasma       S 2.22   S 3.0    S 4.0  S 2.19                                  n = 17       n = 22   n = 4    n = 4  n = 23                                  ______________________________________                                        pH      7.31     7.14     7.12   7.13   7.04                                  Agg     82       83       76     78     81                                    GMP-140 52       49       46     45     68                                    ______________________________________                                         *No UVA; Day 1 of Storage                                                

                  TABLE 24*                                                       ______________________________________                                        Plasma       S 2.22   S 3.0    S 4.0  S 2.19                                  n = 18       n = 20   n = 4    n = 4  n = 23                                  ______________________________________                                        pH      7.03     6.92     6.93   6.93   6.96                                  Agg     75       70       67     70     64                                    GMP-140 61       63       63     64     74                                    ______________________________________                                         *No UVA; Day 5 of Storage                                                

                  TABLE 25*                                                       ______________________________________                                                S 2.22                                                                              S 3.0      S 4.0   S 2.19                                               n = 20                                                                              n = 4      n = 4   n = 22                                       ______________________________________                                        pH        6.80    6.78       6.79  6.95                                       Agg       59      54         54    58                                         GMP-140   73      76         76    83                                         ______________________________________                                         *3 Joules UVA; Day 5 of Storage                                          

EXAMPLE 23

In this example, bacterial inactivation efficiency of several compoundsof the present invention was evaluated by examining the ability of thecompounds to inactivate a variety of bacteria. For this experiment,twelve phylogenetically distinct and clinically pathogenic strains ofbacteria were studied. Table 26 contains a list of these bacteria. Allbacteria were obtained from the Department of Public Health Services inBerkeley, Calif. Inactivation assays were performed as follows.

                  TABLE 26                                                        ______________________________________                                        Corynebacterium minutissimum                                                                    gram positive                                                                             Public Health                                                                 #88A-1157                                       Enterobacter cloacae                                                                            gram negative                                                                             Public Health                                                                 #6710-A-76                                      Escherichia coli  gram positive                                                                             Public Health                                                                 #7186-75                                        Klebsiella pneumoniae                                                                           gram negative                                                                             Public Health                                                                 #92A-2214                                       Listeria monocytogenes                                                                          gram positive                                                                             Public Health                                                                 #86A-6620                                       Pseudomonas aeruginosa                                                                          gram negative                                                                             Public Health                                                                 #91A-5818                                       Salmonella cholerasius                                                                          gram negative                                                                             Public Health                                                                 #92A-2467                                       Serratia marcescens                                                                             gram negative                                                                             Public Health                                                                 #2574-3-79                                      Staphylococcus aureus                                                                           gram positive                                                                             Public Health                                                                 #89A-3667                                       Staphylococcus epidermidis                                                                      gram positive                                                                             Public Health                                                                 #85A-2460                                       Streptococcus Group A                                                                           gram positive                                                                             Public Health                                                                 #90A-2540                                       Yersinia enterocolitica                                                                         gram positive                                                                             Public Health                                                                 #91A-7632                                       ______________________________________                                    

For E. cloacae, E. coli, K pneumoniae, L. monocytogenes, P. aeruginosa,S. cholerasius, S. marcescens, S. aureus, S. epidermidis and Y.enterocolitica, a single colony of bacteria was taken from Luria-Bertani(LB) agar plates [LB broth-Bactotryptone 10 g/L (Difco, Detroit Mich.),Yeast extract 5 g/L, NaCl 10 g/L; LB Agar plates-LB and Bacto-agar 15g/L (Difco) in petri dish] and inoculated in to 3 ml LB broth in aFalcon 2051 capped tube (Becton Dickinson, Rutherford, N.J.). Bacteriawere grown overnight in a Lab Line Orbit Environ-shaker (Labline,Melrose Park, Ill.) at 37° C.

For C. minutissimum, L. monocytogenes and S. Group A, bacteria werestreaked on TSA II Soy Agar with 5% Sheep blood plates (BBL stackerplate; Becton Dickinson, Cockeysville, Md.). Plates were incubatedovernight at 37° C. Then 2.5 ml of LB broth were added to the plate.Colonies were scraped into the broth using a sterile loop. LB brothcontaining the bacteria was then removed from the plate into a sterileFalcon 2051 culture tube.

For all bacteria strains, 100 ml of bacteria suspension were dilutedwith 900 ml of LB broth. Using LB broth as a reference, a spectrum wastaken from 400-800 nm using a Shimadzu UV160U spectrophotometer(Shimadzu, Kyoto, Japan). Bacterial density was calculated assuming thatan optical density (OD) of 1.0 at 600 nm was equivalent to 5×10⁸ cfu/ml(colony forming units/ml). OD readings ranged from 1.1×10⁹ cfu/ml to2.75×10⁹ cfu/ml.

For S. cholcrasius, E. cloacae, K. pneumoniae and P. aeruginosa,recently outdated platelet concentrate units from a local Blood Bankwere stored in Helmer platelet incubator and shaker at 22° C. until use.Two units (each comprising 4 pooled platelet concentrates) ofapproximately 50 ml each were pooled using a Haemonetics SCD 312 steriledocker (Terumo; Braintree, Mass.) in order to have sufficient volume foreach experiment. The platelet concentrate was divided evenly in 50 mlconical centrifuge tubes (Starstedt). Tubes were spun at 3800 rpm for 6minutes with brake 6 on a Sorvall RC3B Centrifuge (DuPont Company;Wilmington, Del.). Plasma was removed and replaced with a syntheticmedia (comprising: 116 mM NaCl, 10 mM Na₃ citrate, 30 mM Na acetate,having pH adjusted to 7.2 with HCl) such that the final concentrationwas 35% plasma 65% synthetic media. Platelets were resuspended viaserial pipette (Falcon, Becton Dickinson; Rutherford, N.J.). For therest of the bacteria, apheresis units were used. These also containedthe same synthetic media, however, S. aureus had additional phosphate(26 mM) and S. Group A, L. monocytogenes had additional phosphate (26mM) and mannitol (20 mM).

Blood gas analysis was done on a Ciba-Corning 238 pH/Blood Gas Analyzer(Ciba-Corning; Medfield, Mass.) and ranged from 6.57 to 7.08 pH.Platelet count was performed by diluting platelet concentrate 1:3 in S/PCertified Blood Bank Saline (Baxter), diluting that sample using aSysmex DD-100 with Quicklyser-II™ (TOA Medical Electronics Co., LTD;Kobe, Japan) and using a Sysmex Microcell counter F-800. Numbers weremultiplied by 3 to get actual concentration of platelets which rangedfrom 8.52×10⁵ /ml to 1.1×10⁷ /ml.

An aliquot (30 ml) of this platelet suspension was put into three 50 mltubes. Bacteria was added to all three such that the final concentrationwas 10⁶ plaque forming units (pfu)/ml based on the count taken above,except P. aeruginosa, which was added at 10⁵ pfu/ml. Two of the threetubes had Compound 2 added at a final concentration of 150 mM. From eachtube, 27 ml was put into mini PL732 blood bags (Baxter Fenwall,Deerfield, Ill.) using a serological pipette and a 20 cc syringe. Asmuch air as possible was removed from the bag before capping. Bagthickness was about 1 cm at the highest point when completely filled.For S. marcescens, K. pneumonae, S. cholerasuis, and E. cloacae, 2additional tubes were set up with 10⁵ cfu/ml bacteria and 150 mMCompound 2. For P. aeruginosa, duplicates were set up for 10⁵ cfu/ml,10⁴ cfu/ml, and 10³ cfu/ml with 150 mM Compound 2.

Bags were laid on their side in the device described in Example 1,above. Irradiation was for one Joule intervals with constant shaking.Between irradiations, bags were picked up and mixed by hand. Allbacteria were irradiated for 2 joules total, except S. marcenscens K.pneumonia, and P. aeruginosa, which were irradiated for 3 joules.

After the two joule irradiation, the exterior of the bag was wiped offusing 70% ethanol. A 1 cc insulin syringe with a 28 guage 1/2 in. needlewas used to remove 2 aliquots from the bag. Approximately 300 ml wasremoved into a sterile Falcon 2017 culture tube for plating on LB agar.The culture tube was gently vortexed and 2 aliquots of 100 ml wasremoved and sterile plated on 2 LB plates. C. minutissimum, L.monocytogenes and S. Group A were plated on Soy Agar with 5% Sheep bloodplates. Plates were incubated overnight at 37° C.

Treated platelet concentrate (1 ml) was incubated in 9 ml of sterile LBin a 50 ml Starstedt tube. The lid was loosely attached with tape.Culture was shaken overnight at 37° C. and was then streaked on sterileLB plates using a sterile loop. C. minutissimum, L. monocytogenes and S.Group A were plated on Soy Agar with 5% Sheep blood plates These plateswere incubated overnight at 37° C. Complete kill was determined by cleanplates after the overnight incubation. The sampling procedure wasrepeated at the 3 joule point for gram negative bacteria samples.

Serial 1:10 dilutions using non-irradiated plateletconcentrate/bacteria/Compound 2 as well as for the irradiated UVAcontrol were done in sterile Falcon 2017 culture tubes containing LBbroth down to 10⁻⁶ dilution. The 10⁻¹ to 10⁻³ dilutions of these werediscarded as colonies would have been too numerous to count. Culturetubes were gently vortexed and 100 ml was sterile plated on LB plates.C. minutissiumum, L. monocytogenes and S. Group A were plated on SoyAgar with 5% Sheep blood plates. Plates were incubated overnight in 37°C. incubator.

Colonies were counted and the numbers obtained were multiplied by theappropriate dilution factor to get cfu/ml. Titers for each treatmentwere averaged and the log₁₀ bacteria was calculated. Log₁₀ kill wascalculated by subtracting the treated bacteria log₁₀ from the untreatedbacteria log₁₀. For samples that showed no colonies, 1 ml samples werethen plated, incubated and counted. If these plates showed no colonies,the kill was considered to be complete.

The results appear in Table 27, below. Complete kill, to the detectionlimit of the assay used, was achieved against all bacteria tested.

                  TABLE 27                                                        ______________________________________                                        Organism         Starting Log Titer                                                                         Log Kill                                        ______________________________________                                        Corynebacterium minutissimum                                                                   5.79         complete kill                                   Enterobacter cloacae                                                                           6.1          complete kill                                   Escherichia coli 6.18         complete kill                                   Klebsiella pneumoniae                                                                          6.58         complete kill                                   Listeria monocytogenes                                                                         6.82         complete kill                                   Pseudomonas aeruginosa                                                                         4.52         complete kill                                   Salmonella cholerasius                                                                         6.22         complete kill                                   Serratia marcescens                                                                            5.32         complete kill                                   Staphylococcus aureus                                                                          6.44         complete kill                                   Staphylococcus epidermidis                                                                     5.8          complete kill                                   Streptococcus Group A                                                                          6.07         complete kill                                   Yersinia enterocolitica                                                                        6.13         complete kill                                   ______________________________________                                    

It is to be understood that the invention is not to be limited to theexact details of operation or exact compounds, composition, methods, orprocedures shown and described, as modifications and equivalents will beapparent to one skilled in the art. All patents described are herebyincorporated by reference.

We claim:
 1. A method of inactivating pathogens in a compositioncomprising a blood product, comprising the steps of:a) contacting one ormore psoralen compounds or salts thereof of formula I, ##STR3## whereinR₁ is --(CH₂)₂ --NH₂, --(CH₂)_(w) --R₂ --(CH₂)_(z) --NH₂, --(CH₂)_(w)--R₂ --(CH₂)_(x) --R₃ --(CH₂)_(z) --NH₂, or --(CH₂)_(w) --R₂ --(CH₂)_(x)--R₃ --(CH₂)_(y) --R₄ --(CH₂)_(z) --NH₂ ;and R₂, R₃, and R₄ areindependently Q or NH₃, and w is a whole number from 1 to 5, x is awhole number from 2 to 5, y is a whole number from 2 to 5, and z is awhole number from 2 to 6; R₅, R₆ and R₇ are independently H and(CH₂)_(v) CH₃, and v is a whole number from 0 to 5 in a concentrationsufficient to inactivate pathogens upon photoactivation, with a bloodproduct suspected of being contaminated with pathogens; and b)photoactivating said compound for a duration and at an intensity and awavelength sufficient to inactivate the pathogens.
 2. The method ofclaim 1, wherein said compoundis4'-(4-amino-2-aza)butyl-4,5',8-trimethylpsoralen,4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen,4'-(2-aminoethyl)-4,5',8-trimethylpsoralen,4'-(5-amino-2-oxa)pentyl-4,5',8-trimethylpsoralen,4'-(5-amino-2-aza)pentyl-4,5'8-trimethylpsoralen,4'-(6-amino-2-aza)hexyl-4,5',8-trimethylpsoralen,4'-(7-amino-2,5-oxa)heptyl-4,5',8-trimethylpsoralen,4'-(12-amino-8-aza-2,5-dioxa)dodecyl-4,5',8-trimethylpsoralen,4'-(13-amino-2-aza-6,11-dioxa)tridecyl-4,5',8-trimethylpsoralen,4'-(7-amino-2-aza)heptyl-4,5',8-trimethylpsoralen,4'-(7-amino-2-aza-5-oxa)heptyl-4,5',8-trimethylpsoralen,4'-(9-amino-2,6-diaza)nonyl-4,5',8-trimethylpsoralen,4'-(8-amino-5-aza-2-oxa)octyl-4,5',8-trimethylpsoralen,4'-(9-amino-5-aza-2-oxa)nonyl-4,5',8-trimethylpsoralen, or4'-(14-amino-2,6,11-triaza)tetradecyl-4,5',8-trimethylpsoralen.
 3. Themethod of claim 1, wherein said pathogen comprises a gram positivebacteria.
 4. The method of claim 1, wherein said pathogen comprises agram negative bacteria.
 5. The method of claim 1, wherein said pathogencomprises a virus.
 6. The method of claim 1, wherein said blood productcomprises platelets.
 7. The method of claim 1, wherein said bloodproduct comprises plasma.
 8. The method of claim 1, wherein saidwavelength is between 180 nm and 400 nm.
 9. The method of claim 1,wherein said wavelength is between 320 nm and 380 nm.
 10. The method ofclaim 8, wherein the intensity is between 1 and 30 mW/cm².
 11. Themethod of claim 8; wherein the duration is between 1 second and thirtyminutes.
 12. The method of claim 1, wherein said blood product furthercomprises a synthetic media.
 13. The method of claim 1, wherein saidcompound is in a final concentration of between 0.1 and 250 μM.
 14. Themethod of claim 13, wherein said compound is in between 10 and 150 μM.15. The method of claim 1, wherein the photoactivating step is performedwithout limiting the concentration of molecular oxygen.
 16. The methodof claim 1, wherein at least two of said compounds are present.
 17. Themethod of claim 1, wherein said compound is in a solution prior tocontacting the blood product.
 18. The method of claim 17, wherein saidsolution comprises water, saline, or a synthetic media.
 19. The methodof claim 18, wherein said synthetic media comprises sodium acetate,potassium chloride, sodium chloride, sodium citrate, sodium phosphateand magnesium chloride.
 20. The method of claim 19, wherein saidsynthetic media further comprises at least one constituent selected fromthe group consisting of mannitol and glucose.
 21. The method of claim18, wherein, said solution is contained in a first blood bag and saidblood product is contained in a second blood bag.
 22. The method ofclaim 21, wherein said contacting is by expressing said solution fromsaid first blood bag into said second blood bag via a sterileconnection.
 23. The method of claim 1, wherein said compound is in a drystate prior to contacting.